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.
Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant.
Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis, a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants.
SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.
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.
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.
Carboxyl/cholinesterases (CCEs) have pivotal roles in dietary detoxification, pheromone or hormone degradation and neurodevelopment. The recent completion of genome projects in various insect species has led to the identification of multiple CCEs with unknown functions. Here, we analyzed the phylogeny, expression and genomic distribution of 69 putative CCEs in the silkworm, Bombyx mori (Lepidoptera: Bombycidae).
A phylogenetic tree of CCEs in B. mori and other lepidopteran species was constructed. The expression pattern of each B. mori CCE was also investigated by a search of an expressed sequence tag (EST) database, and the relationship between phylogeny and expression was analyzed. A large number of B. mori CCEs were identified from a midgut EST library. CCEs expressed in the midgut formed a cluster in the phylogenetic tree that included not only B. mori genes but also those of other lepidopteran species. The silkworm, and possibly also other lepidopteran species, has a large number of CCEs, and this might be a consequence of the large cluster of midgut CCEs. Investigation of intron-exon organization in B. mori CCEs revealed that their positions and splicing site phases were strongly conserved. Several B. mori CCEs, including juvenile hormone esterase, not only showed clustering in the phylogenetic tree but were also closely located on silkworm chromosomes. We investigated the phylogeny and microsynteny of neuroligins in detail, among many CCEs. Interestingly, we found the evolution of this gene appeared not to be conserved between B. mori and other insect orders.
We analyzed 69 putative CCEs from B. mori. Comparison of these CCEs with other lepidopteran CCEs indicated that they had conserved expression and function in this insect order. The analyses showed that CCEs were unevenly distributed across the genome of B. mori and suggested that neuroligins may have a distinct evolutionary history from other insect order. It is possible that such an uneven genomic distribution and a unique neuroligin evolution are shared with other lepidopteran insects. Our genomic analysis has provided novel information on the CCEs of the silkworm, which will be of value to understanding the biology, physiology and evolution of insect CCEs.
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.
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.
insect; silkmoth; olfaction; sex pheromone; pheromone-source searching behavior
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.
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction
Trifluoromethyl ketones reversibly inhibit pheromone-degrading esterases in insect olfactory tissues, affecting pheromone detection and behavior of moth males. In this work, (Z)-9-tetradecenyl trifluoromethyl ketone (Z9-14:TFMK), a closely-related analogue of the pheromone of the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), was prepared and tested in electroantennogram and field tests as possible inhibitors of the pheromone action. The electroantennogram parameters, amplitude, and the repolarization time of the antennal responses of S. frugiperda males were affected by Z9-14:TFMK vapors. Exposure of male antennae to a stream of air passing through 100 ìg of the ketone produced a significant reduction of the amplitude and an increase of 2/3 repolarization time signals to the pheromone. The effect was reversible and dose-dependent. In the field, the analogue significantly decreased the number of males caught when mixed with the pheromone in 10:1 ratio. The results suggest that Z9-14:TFMK is a mating disruptant of S. frugiperda and may be a good candidate to consider in future strategies to control this pest.
pheromone inhibition; mating disruptant; antagonism; trifluoromethyl ketones
Chemosensory receptors including olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) play a central role in sensing chemical signals and guiding insect behaviours, and are potential target genes in insect pest control. The cotton bollworm Helicoverpa armigera is one of the most destructive pest species that can feed on over 200 different plant species. This diversity of host plants is likely linked to a complex chemosensory system. Here we built on previous work to characterize crucial chemosensory tissues linked to environmental interactions including larval antennae, larval mouthparts and larval fat bodies, as well as male and female adult heads, male and female adult tarsi, and female abdomens.
Using transcriptome sequencing, Trinity RNA-seq assemblies and extensive manual curation, we identified a total of 91 candidate chemosensory receptors (60 candidate ORs, 10 GRs and 21 IRs). Thirty-five of these candidates present full-length transcripts. First, we performed in silico differential expression analysis on different sequenced tissues. Further, we created extensive expression profiles using reverse transcription (RT)-PCR on a variety of adult and larval stages. We found that the expression profile of HarmOR51 was limited to adult male antenna suggesting a role in mating that was further supported by a phylogenetic analysis clustering it into the pheromone receptor clade. HarmOR51 in calcium imaging analysis did not show responses to either of the two H. armigera sex pheromone components (Z9-16:Ald or Z11-16:Ald) inviting a future detailed study. In addition, we found four novel HarmORs (OR1, 53, 54 and 58) that appeared to be larvae-antennal specific. Finally, our expression profiling showed that four “divergent” HarmIRs (IR2, 7d.1, 7d.2 and 7d.3) were expressed in both adult and larval antennae, suggesting a functional divergence from their Drosophila homologues.
This study explored three chemoreceptor superfamily genes using a curated transcriptomic approach coupled with extensive expression profiling and a more limited functional characterization. Our results have now provided an extensive resource for investigating the chemoreceptor complement of this insect pest, and meanwhile allow for targeted experiments to identify potential molecular targets for pest control and to investigate insect-plant interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-597) contains supplementary material, which is available to authorized users.
Helicoverpa armigera; Olfactory receptor; Gustatory receptor; Ionotropic receptor; Expression profile
The males of some species of moths possess elaborate feathery antennae. It is widely assumed that these striking morphological features have evolved through selection for males with greater sensitivity to the female sex pheromone, which is typically released in minute quantities. Accordingly, females of species in which males have elaborate (i.e., pectinate, bipectinate, or quadripectinate) antennae should produce the smallest quantities of pheromone. Alternatively, antennal morphology may be associated with the chemical properties of the pheromone components, with elaborate antennae being associated with pheromones that diffuse more quickly (i.e., have lower molecular weights). Finally, antennal morphology may reflect population structure, with low population abundance selecting for higher sensitivity and hence more elaborate antennae. We conducted a phylogenetic comparative analysis to test these explanations using pheromone chemical data and trapping data for 152 moth species. Elaborate antennae are associated with larger body size (longer forewing length), which suggests a biological cost that smaller moth species cannot bear. Body size is also positively correlated with pheromone titre and negatively correlated with population abundance (estimated by male abundance). Removing the effects of body size revealed no association between the shape of antennae and either pheromone titre, male abundance, or mean molecular weight of the pheromone components. However, among species with elaborate antennae, longer antennae were typically associated with lower male abundances and pheromone compounds with lower molecular weight, suggesting that male distribution and a more rapidly diffusing female sex pheromone may influence the size but not the general shape of male antennae.
Antennal morphology; forewing length; Lepidoptera; phylogenetic generalized least squares; sex pheromone
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.
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.
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.
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.
Electrophysiology; Glomerulus; Neural coding; Olfaction; Pheromone
In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca2+) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca2+ concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca2+ stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca2+ pump in the secretory pathway, exhibit higher rates of Ca2+ influx relative to wild-type cells due to the stimulation of a high-affinity Ca2+ uptake system. Stimulation of this Ca2+ uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca2+ uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca2+ transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca2+ uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca2+ influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca2+ are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca2+ channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.
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.
There is growing recognition of the importance of the active involvement of consumers and community members in health care. Despite the long history of consumer and community engagement (CCE) research and practice, there is no consensus on the best strategies for CCE. In this paper, we identify various dimensions of CCE-related strategies and offer a practical model to assist policy-makers, practitioners and researchers.
We undertook a large-scale, scoping meta-review and searched six databases using a list of nine medical subject headings (MeSH) and a comprehensive list of 47 phrases. We identified and examined a total of 90 relevant systematic reviews.
Identified reviews show that although there is a significant body of research on CCE, the development of the field is hindered by a lack of evidence relating to specific elements of CCE. They also indicate a diverse and growing enterprise, drawing on a wide range of disciplinary, political and philosophical perspectives and a mix of definitions, targets, approaches, strategies and mechanisms. CCE interventions and strategies aim to involve consumers, community members and the public in general, as well as specific sub-groups, including children and people from culturally and linguistically diverse backgrounds. Strategies for CCE vary in terms of their aim and type of proposed activity, as do the methods and tools which have been developed to support them. Methods and tools include shared decision making, use of decision aids, consumer representation, application of electronic and internet-based facilities, and peer support. The success of CCE is dependent on both the approach taken and contextual factors, including structural facilitators such as governmental support, as well as barriers such as costs, organisational culture and population-specific limitations.
The diversity of the field indicates the need to measure each component of CCE. This meta-review provides the basis for development of a new eight stage model of consumer and community engagement. This model emphasises the importance of clarity and focus, as well as an extensive evaluation of contextual factors within specific settings, before the implementation of CCE strategies, enabling those involved in CCE to determine potential facilitators and barriers to the process.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6963-14-402) contains supplementary material, which is available to authorized users.
Consumer and community engagement (CCE); Shared decision making (SDM); Consumer representation; Patient involvement; Implementation
In the course of evolution butterflies and moths developed two different reproductive behaviors. Whereas butterflies rely on visual stimuli for mate location, moths use the ‘female calling plus male seduction’ system, in which females release long-range sex pheromones to attract conspecific males. There are few exceptions from this pattern but in all cases known female moths possess sex pheromone glands which apparently have been lost in female butterflies. In the day-flying moth family Castniidae (“butterfly-moths”), which includes some important crop pests, no pheromones have been found so far.
Using a multidisciplinary approach we described the steps involved in the courtship of P. archon, showing that visual cues are the only ones used for mate location; showed that the morphology and fine structure of the antennae of this moth are strikingly similar to those of butterflies, with male sensilla apparently not suited to detect female-released long range pheromones; showed that its females lack pheromone-producing glands, and identified three compounds as putative male sex pheromone (MSP) components of P. archon, released from the proximal halves of male forewings and hindwings.
This study provides evidence for the first time in Lepidoptera that females of a moth do not produce any pheromone to attract males, and that mate location is achieved only visually by patrolling males, which may release a pheromone at short distance, putatively a mixture of Z,E-farnesal, E,E-farnesal, and (E,Z)-2,13-octadecadienol. The outlined behavior, long thought to be unique to butterflies, is likely to be widespread in Castniidae implying a novel, unparalleled butterfly-like reproductive behavior in moths. This will also have practical implications in applied entomology since it signifies that the monitoring/control of castniid pests should not be based on the use of female-produced pheromones, as it is usually done in many moths.
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.
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.
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.
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.
Pheromone binding proteins; General odorant binding proteins; Real-time PCR; Fluorescence competitive binding assay; Forest insect; Economic pest; Lepidoptera
In the olfactory pathway of Drosophila, a GABAB receptor mediated presynaptic gain control mechanism at the first synapse between olfactory sensory neurons (OSNs) and projection neurons has been suggested to play a critical role in setting the sensitivity and detection range of the sensory system. To approach the question if such a mechanism may be realized in the pheromone recognition system of male moths in this study attempts were made to explore if moth's pheromone-responsive cells express a GABAB- receptor. Employing a combination of genome analysis, RT-PCR experiments and screening of an antennal cDNA library we have identified a cDNA which encodes the GABAB-R1 receptor of Heliothis virescens. Moreover, based on the HvirGABAB-R1 sequence we could predict a GABAB-R1 protein from genome sequences of the silkmoth Bombyx mori. To assess whether HvirGABAB-R1 is expressed in OSNs of male antenna we performed whole-mount in situ hybridization (WM-ISH) experiments. Several HvirGABAB-R1 positive cells were visualized under long sensilla trichodea, known to contain pheromone-responsive OSNs. In parallel it was shown that cells under long trichoid hairs were labelled with pheromone receptor specific probes. In addition, the HvirGABAB-R1 specific probe also labelled several cells under shorter olfactory sensilla, but never stained cells under mechanosensory/gustatory sensilla chaetica. Together, the results indicate that a GABAB receptor is expressed in pheromone-responsive OSNs of H. virescens and suggest a presynaptic gain control mechanism in the axon terminals of these cells.
moth; olfaction; GABA; pheromone; in situ hybridization.
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.
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.
Tremendous evolutional success and the ecological dominance of social insects, including ants, termites and social bees, are due to their efficient social organizations and their underlying communication systems. Functional division into reproductive and sterile castes, cooperation in defending the nest, rearing the young and gathering food are all regulated by communication by means of various kinds of pheromones. No brain structures specifically involved in the processing of non-sexual pheromone have been physiologically identified in any social insects. By use of intracellular recording and staining techniques, we studied responses of projection neurons of the antennal lobe (primary olfactory centre) of ants to alarm pheromone, which plays predominant roles in colony defence. Among 23 alarm pheromone-sensitive projection neurons recorded and stained in this study, eight were uniglomerular projection neurons with dendrites in one glomerulus, a structural unit of the antennal lobe, and the remaining 15 were multiglomerular projection neurons with dendrites in multiple glomeruli. Notably, all alarm pheromone-sensitive uniglomerular projection neurons had dendrites in one of five ‘alarm pheromone-sensitive (AS)’ glomeruli that form a cluster in the dorsalmost part of the antennal lobe. All alarm pheromone-sensitive multiglomerular projection neurons had dendrites in some of the AS glomeruli as well as in glomeruli in the anterodorsal area of the antennal lobe. The results suggest that components of alarm pheromone are processed in a specific cluster of glomeruli in the antennal lobe of ants.
social insects; alarm pheromone; antennal lobe; olfactory centre; pheromone communication
The relative proportions of components in a pheromone blend play a major role in sexual recognition in moths. Two sympatric species, Helicoverpa armigera and Helicoverpa assulta, use (Z)-11-hexadecenal (Z11–16: Ald) and (Z)-9-hexadecenal (Z9–16: Ald) as essential sex pheromone components but in very different ratios, 97∶3 and 7∶93 respectively. Using wind tunnel tests, single sensillum recording and in vivo calcium imaging, we comparatively studied behavioral responses and physiological activities at the level of antennal sensilla and antennal lobe (AL) in males of the two species to blends of the two pheromone components in different ratios (100∶0, 97∶3, 50∶50, 7∶93, 0∶100). Z11–16: Ald and Z9–16: Ald were recognized by two populations of olfactory sensory neurons (OSNs) in different trichoid sensilla on antennae of both species. The ratios of OSNs responding to Z11–16:Ald and Z9–16:Ald OSNs were 100∶28.9 and 21.9∶100 in H. armigera and H. assulta, respectively. The Z11–16:Ald OSNs in H. armigera exhibited higher sensitivity and efficacy than those in H. assulta, while the Z9–16:Ald OSNs in H. armigera had the same sensitivity but lower efficacy than those in H. assulta. At the dosage of 10 µg, Z11–16: Ald and Z9–16: Ald evoked calcium activity in 8.5% and 3.0% of the AL surface in H. armigera, while 5.4% and 8.6% of AL in H. assulta, respectively. The calcium activities in the AL reflected the peripheral input signals of the binary pheromone mixtures and correlated with the behavioral output. These results demonstrate that the binary pheromone blends were precisely coded by the firing frequency of individual OSNs tuned to Z11–16: Ald or Z9–16: Ald, as well as their population sizes. Such information was then accurately reported to ALs of H. armigera and H. assulta, eventually producing different behaviors.