Chemical constraints on the evolution of olfactory communication channels of moths

It is estimated there are over 100,000 moth species (Lepidoptera) that produce sex pheromones comprising communication channels used in specific-mate-recognition systems (SMRS) involved in pre-zygotic mating isolation and speciation. About 1572 moth species have been found to use 377 pheromone components, the majority being alcohols, aldehydes or acetate esters of olefinic chains of 10-22 carbons. Since there are limited numbers of possible unsaturated (double bonded) E- and Z-configured isomers of these chains, there may be constraints on incipient species evolving new pheromone components, especially if they are utilized by existing species. Here I develop algorithms that count and name all possible structures of chains with or without a functional group. The results show that for acetate esters there are only seven or nine monounsaturated isomers of six or seven carbon chains, respectively, suggesting use of these compounds could limit the number of communication channels available for radiation of new species (no moths use these short chains). For commonly used 14-carbon chains with an acetate ester functional group, and 1-3 unsaturations, there are 1039 isomers. A total of 2,096,883 isomers were counted from all multiply unsaturated 10-22-carbon chain acetate esters. The number of possible signals quickly extends into millions when considering pheromone blends of 2-4 components used by most species. There should be little chemical constraint on evolution of new communication channels based on compounds of 10 or more carbons, even for closely related species (e.g. ermine moths, Yponomeuta).     

Feeding and hemolymph trehalose concentration influence sex pheromone production in virgin Heliothis virescens moths

Previously, we demonstrated that sex pheromone production in mated female Heliothis virescens moths is dependent upon hemolymph trehalose concentration (HTC), which is influenced by activities such as the feeding of adults on sucrose. In this paper we demonstrate, for the first time, that this effect also occurs in starved (i.e., sugar-stressed) virgin females. Females allowed to feed on sugar for 6 days, following eclosion, had significantly greater titers than females that had fed only on water (i.e., were starved). No differences in pheromone titer were observed between sugar- and water-fed females at shorter (1 or 3 days) periods following eclosion. The relatively short-term effects of HTC on sex pheromone titer of virgins, were demonstrated by feeding experiments, in which starved (for 4 days) virgins fed on 10% sucrose solution had significantly greater HTC and pheromone titers than ones fed only on water; an increase in HTC was apparent within an hour, while the increase in pheromone titer was apparent within 2.5 h, of sugar feeding. Starvation also showed similar effects on titers of pheromone gland fatty acids (pheromone intermediates) and HTC. Over 6 days of starvation, fatty acid titers and HTC declined gradually. After feeding on sucrose, titers of hexadecanoic, (Z)-9-hexadecanoic, (Z)-11-hexadecanoic and (Z)-9-octadecanoic, acids, as well as HTC, increased significantly 24 h later, but titers of octadecanoic and (Z,Z)-9,12-octadecanoic (linoleic) acids did not. Lepidoptera cannot biosynthesize polyunsaturated acids, but the lack of change in octadecanoic acid titer suggests this acid may not participate in pheromone biosynthesis. In addition to these short-term changes in pheromone and fatty acid production, mediated by HTC, a longer-term effect of age, regardless of HTC, on pheromone titer was observed. Overall, these results are consistent with hemolymph trehalose and glandular fatty acids acting as twin metabolite reservoirs for pheromone biosynthesis. Hemolymph trehalose, able to be refilled through feeding on exogenous sugars, has a one-way flow of metabolites for synthesis of glandular free fatty acids (FFAs) and pheromone, while glandular glycerolipids provide a reversible reservoir for metabolites, accepting surplus FFAs when glandular concentrations are high, and providing FFAs for pheromone biosynthesis when concentrations are low.     

Physiology of interspecific chemical communication in Heliothis moths

Abstract Electroantennograms were recorded from the antennae of adult male and female corn earworms, Heliothis zea (Boddie). A total of seventeen female moth sex pheromone components from several species were tested. Of these, two components elicited significantly greater responses than the other fifteen. These were (Z)-11-hexadecenal, a conspecific component, and (Z)-9-tetradecenal, a component found in the pheromone blend of a sympatric species H.virescens (F.) that inhibits attraction of H.zea males. The results from dose-response and selective adaptation studies indicate that there are separate populations of receptors for these two chemical signals on the antenna of male H.zea. The more sensitive population is selective for (Z)-11-hexadecenal, while the less sensitive one responds to (Z)-9-tetradecenal. These findings provide a physiological basis by which H.zea males can distinguish the interspecific repellent from the conspecific pheromone blend. It is likely that this discrimination contributes to reproductive isolation between these two species.     

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

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

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

1H, 15N, and 13C Chemical shift assignments of the navel orange worm pheromone-binding protein-1 (Atra-PBP1)

A pheromone-binding protein from navel orange worm, Amyelois transitella (Atra-PBP1) binds to non-polar pheromone molecules and facilitates the transport and delivery of pheromone to the membrane-bound pheromone receptors. We report complete NMR chemical shift assignments of Atra-PBP1 obtained at pH 4.5 and 25°C (BMRB No. 15601).     

Chemical communication in heliothine moths. VII. Correlation between diminished responses to point-source plumes and single filaments similarly tainted with a behavioral antagonist

Addition of (Z)-11-hexadecenyl acetate (Z11-16:Ac) into a normally attractive binary blend of Heliothis virescens pheromone components resulted in a suppression of upwind flight and source location by males. Male response was reduced even at the lowest dosages of Z11-16:Ac tested but upwind flight and source location were most clearly reduced when the loading of Z11-16:Ac reached 10% or more of the (Z)-11-hexadecenal (Z11-16:Ald) loading (the major component present in the binary blend). Similar patterns of suppression in response were noted when Z11-16:Ac was added to binary blends of pheromone components at both 10 and 100 μg loadings of Z11-16:Ald. Males in casting flight following upwind flight in a mechanically generated pulsed plume, responded to the interception of a subsequent, single binary-blend filament by making a toward-source upwind surge. Responses of males to a single filament that was tainted by a level of Z11-16:Ac that had allowed some reduced level of upwind flight and source location to occur in the previous plume experiments were diminished compared with their control counterparts. Analysis of the flight tracks revealed that the surges in response to single tainted filaments were stunted because males made fewer significant changes in course angles steered, airspeeds generated, and in the tempo of counterturns executed. Accepted: 28 December 1996     

Flight speed of male spruce bud worm moths in a wind tunnel at different wind speeds and at different distances from a pheromone source

ABSTRACT. Flying male spruce budworm ( Choristoneura fumiferana [Clem.]) moths responding to virgin females and to synthetic pheromone in a wind tunnel maintained a constant rate of upwind progress when held by moving optomotor cues at a constant distance from the pheromone source. When allowed to progress upwind to the source, however, they slowed their upwind speed progressively as they approached it. They also adjusted their flight speed to maintain similar rates of upwind progress at different wind speeds.     

Mice (Mus musculus) lacking a vomeronasal organ can discriminate MHC-determined odortypes

Major histocompatibility complex (MHC) genes in mammals (H-2 in mice) play a major role in regulating immune function. They also bestow individuality in the form of a chemical signature or odortype. At present, the respective contributions of the olfactory epithelium and the vomeronasal organ (VNO) in the recognition of individual odortypes are not well defined. We examined a possible role for the VNO in the recognition of MHC odortypes in mice by first removing the organ (VNX) and then training the mice to distinguish the odors of two congenic strains of mice that differed only in their MHC type. C57BL/6J mice (bb at H-2) and C57BL/6J-H-2(k) (kk at H-2) provided urine for sensory testing. Eight VNX and six sham-operated mice were trained to make the discrimination. Neither the number of training trials-to-criterion nor the rate of learning differed significantly for VNX and sham-operated mice. We conclude that the VNO is not necessary for learning to discriminate between MHC odortypes.     

Structure and function of antennal lobe neurons in the male turnip moth,Agrotis segetum (Lepidoptera: Noctuidae)

Interneurons with dendritic branches in the antennal lobe of the male turnip moth, Agrotis segetum (Schiff., Lepidoptera: Noctuidae), were investigated with intracellular recording and staining methods. Seventeen projection neurons that transmit information from the antennal lobe to higher centers in the brain displayed dendritic arbors in the male specific macroglomerular complex (MGC) and responded to chemical components of the female sex pheromone used in species-specific sexual communication. Most of the projection neurons responded to several of the pheromone components tested, and a precise correlation between the location of the dendritic arborization and the physiological response could not be demonstrated. One MGC-projection neuron fit the definition of blend specialist. It did not respond to the individual components of the behaviorally active pheromone blend, but showed a strong response to the components when combined in the species-specific blend. Some of the projection neurons also showed clear responses to phenylacetaldehyde, a flower-produced compound and/or to (E)-2-hexenal, a common green-leaf volatile. In eight neurons, the axonal projection could be followed to the calyces of the mushroom body, and subsequently to the inferior lateral protocerebrum.Four local interneurons were characterized both morphologically and physiologically. Each neuron arborized extensively throughout the antennal lobe, and each responded to one or several of the pheromone compounds, and/or to one or both of the plant-produced compounds. One of the local interneurons responded exclusively to the pheromone blend, but not to the individual components.Abbreviations AL antennal lobe - AN antennal nerve - CB cell body - E2H (E)-2-hexenal - IACT inner antennocerebral tract - ILPR inferior lateral protocerebrum - LH lateral horn of the protocerebrum - LN local interneuron - MB mushroom body - MGC macroglomerular complex - OACT outer antennocerebral tract - PAA phenylacetaldehyde - PN projection interneuron - RN receptor neuron - Z5-10:OAc (Z)-5-decenyl acetate - Z5-10:OH (Z)-5-decenol - Z5-12:OAc (Z)-5-dodecenyl acetate - Z7-12:OAc (Z)-7-dodecenyl acetate - Z9-14:OAc (Z)-9-tetradecenyl acetate     

啮齿动物的嗅觉通讯研究进展

通过对近40 年来啮齿动物嗅觉通讯的研究综述, 主要介绍嗅觉信号的来源、组成及其对啮齿动物行为生理所产生的作用。啮齿动物嗅觉通讯的信号来源主要是粪便、尿液和特化皮肤腺等, 对这些化学信号的成分分析主要集中在各种信息素(Pheromone) 的结构、来源及其引起的行为反应。目前, 在对啮齿动物嗅觉通讯神经通路的研究中, 对主嗅觉系统和犁鼻器系统在动物嗅觉通讯中的作用仍将是人们研究的重点; 而通过信息素作用所产生的各种行为反应的神经内分泌机制也是动物嗅觉通讯领域研究的热点之一。研究气味信号对动物行为和生理等方面所产生的作用, 将有助于揭示啮齿动物嗅觉通讯在其社会行为中的重要作用。     

Activity modulation in cockroach sensillum: the role of octopamine

The plasticity of sensory perception is provided partially by modulation of receptor cells. The electrical activity of American cockroach chemoreceptor cells in response to sex pheromone was measured under the influence of octopamine treatment and tracheal anoxia. Both experimental procedures caused decreased electroantennograms but affected spike activity differently: octopamine treatment increased firing rate, whereas anoxia decreased it. Spike frequency under octopamine treatment was elevated in response to pheromone stimulation and at background activity. Experiments with perfusion of isolated antennae showed a direct effect of octopamine on spike activity of pheromone sensilla, and excluded the possibility of indirect effects via octopamine-dependent release of other biologically active substances. The suggested mechanism of octopamine action is receptor cell membrane depolarization.     

Rate of pheromone release by individual spruce budworm moths

The rate and temporal pattern of pheromone emission by single and grouped female spruce budworm moths were measured by combining the trapping of pheromone on Porapak Q with a rapid luciferase bioassay developed for aldehyde pheromone. Pheromone release occurred mainly during the scotophase of a 12:12 L:D cycle in a series of bursts (up to 50 ng/hr) with considerable variability observed between insects. Analysis of over 30 individual females showed that 30% release no detectable pheromone, 60% release between 20 and 100 ng of pheromone and 10% release greater than 100 ng in a 24-hr period. Overall, a single female (1–3 days old) released an average of 60 ± 50 ng (± S.D.) of pheromone per night with a total of 260 ± 210 ng (± S.D.) being released over its life span (～7 days). Grouped females released lower quantities of pheromone. The amount of pheromone in the glands of female moths also displayed a rhythm with the levels beings higher later in the day than at the start of the photophase.     

Responses of olfactory receptor neurons in Utetheisa ornatrix to gender-specific odors

We recorded the electrophysiological responses of individual olfactory receptor neurons in sensilla trichodea on the antennae of adult arctiid moths, Utetheisa ornatrix, to stimulation with volatiles associated with both sexes. All trichoid sensilla contain at least two receptor neurons, each with distinct action potential amplitudes and waveforms, that respond dichotomously to male and female odors. Although, neither female neuron responds to extracts of coremata or the male-produced pheromone hydroxydanaidal, they do respond in a gender-specific manner to the volatiles emanating from whole pupae, hemolymph, thoracic froth, and adult animals of several ages. Thoracic froth, which contains pyrrolizidine alkaloids, is thought to play a role in defense. Froth from moths reared on diets, with or without added pyrrolizidine alkaloids, were equally effective in eliciting gender-specific patterns of response. Male trichoid receptor neurons respond to these same materials with similar patterns of activation. These receptor neurons provide information about substances, which we have termed “gender odors,” that are persistently emitted by nearby animals. These substances do not appear to be the same as those already known to be involved in defense or the sexual dialog between individuals. Accepted: 16 March 2000     

Pheromone-evoked potentials and oscillations in the antennal lobes of the sphinx moth Manduca sexta

Using intra- and extracellular recording methods, we studied the activity of pheromone-responsive projection neurons in the antennal lobe of the moth Manduca sexta. Intracellularly recorded responses of neurons to antennal stimulation with the pheromone blend characteristically included both inhibitory and excitatory stages of various strengths. To observe the activity of larger groups of neurons, we recorded responses extracellularly in the macroglomerular complex of the antennal lobe. The macroglomerular complex is part of a specialized olfactory subsystem and the site of first-order central processing of sex-pheromonal information. Odors such as the pheromone blend and host-plant (tobacco) volatiles gave rise to evoked potentials that were reproducible upon repeated antennal stimulation. Evoked potentials showed overriding high-frequency oscillations when the antenna was stimulated with the pheromone blend or with either one of the two key pheromone components. The frequency of the oscillations was in the range of 30–50 Hz. Amplitude and frequency of the oscillations varied during the response to pheromonal stimulation. Recording intracellular and extracellular activity simultaneously revealed phase-locking of action potentials to potential oscillations. The results suggest that the activity of neurons of the macroglomerular complex was temporally synchronized, potentially to strengthen the pheromone signal and to improve olfactory perception. Accepted: 19 December 1997     

The role of heliothine hairpencil compounds in female Heliothis virescens (Lepidoptera: Noctuidae) behavior and mate acceptance

Studies on numerous insect species suggest that male-produced sex pheromones play a role in attracting females; as aphrodisiacs, making females more quiescent; or as a means of inhibiting competing males. Male heliothine moths display abdominal hairpencils during courtship, but the specific effects of the odors released on female behavior have not yet been elucidated. This study investigates the role of male hairpencil compounds in female Heliothis virescens mating behavior. Female H. virescens were exposed to filter paper loaded with hairpencil extracts of male H. virescens, Heliothis subflexa and Helicoverpa zea, and observed for behavioral responses to odors. Single synthetic compounds found in the H. virescens hairpencil blend were also tested. In mating assays between single male and female H. virescens it was found that: (i) antennectomized females mated less frequently than sham-operated females; (ii) females mated less frequently with males whose hairpencils had been surgically removed; (iii) females mated with males with ablated hairpencils if a filter paper loaded with one male equivalent of H. virescens hairpencil extract was presented simultaneously; and (iv) this effect was species-specific, as presentation of H. subflexa or H. zea hairpencil extracts did not restore mate acceptance. This study suggests that odors released by male hairpencils are important in mate acceptance by female H. virescens, and may play a role in mate choice and species isolation.     

Optomotor anemotaxis polarizes self-steered zigzagging in flying moths

ABSTRACT. Experiments with oriental fruit moth males, Grapholita molesta (Buck), provide evidence that a pheromone plume in zero wind elicits an endogenous, self-steered programme of counterturning (zigzagging) flight, and that wind experienced in flight establishes the polarity of the counterturns; they become aligned so that displacement occurs toward the source, even after the wind is stopped. In zero wind, males located a pheromone source more frequently when they had experienced a wind after having already taken flight before the wind was stopped (46%) compared with those that took flight later and therefore only experienced wind while they were in contact with the ground (14%). Furthermore, males placed in a stationary pheromone plume in zero wind located the source, eventually, on 21% of occasions. The flight tracks of these males, as well as those having experienced a wind only while on the ground, often exhibited repetitive counterturns (zigzags) of c. 180–200. However, the counterturns meandered around the flight tunnel, the inter-reversal track angles having no consistent direction. Sometimes the males displaced down-tunnel in the stationary plume, sometimes up, eventually locating the source and performing a courtship display. The inter-reversal track angles of males counterturning in wind, on the other hand, displayed a consistent orientation of c. 60 to either side of the wind line, resulting in consistent upwind displacement toward the source. With no pheromone present, with or without wind, counterturns were not observed.