Quantitation of the insect electroantennogram: Measurement of sensillar contributions,elimination of background potentials,and relationship to olfactory sensation

A quantitative study of the electroantennogram (EAG) of Trichoplusia ni (Hübner) response to sex pheromone has provided new information about the physiological basis of the EAG and its relationship to behaviour. Procedures were developed that selectively stimulated defined numbers of sensilla and eliminated extraneous olfactory stimuli. The results show that the EAG is linearly proportional to the number of antennal sensilla stimulated and clarify interpretation of the blank response. Studies with calibrated dispensers indicate that the EAG is related to pheromone concentration by a power function frequently associated with quantitative measures of electrophysiological and behavioural responses in other animals. Based on correlations between the EAG, behaviour, and single-neurone responses, a model is presented that interrelates these three parameters as different indicators of a single quantitative process in the central nervous system.     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

Spectral responses of the tsetse fly, Glossina morsitans morsitans

Spectral responses of male Glossina morsitans morsitans Westw, were investigated using behavioural and electrophysiological techniques. Phototactic responses to monochromatic lights of different wavelengths but equivalent intensities (measured either in energy or quantal units) were tested in an apparatus which permitted the simultaneous presentation of pairs of lights to groups of flies. Near ultraviolet light was the most attractive, followed by blue and red, with green and far red light giving no significant responses above control levels. The spectral sensitivity of the compound eye was also determined from the electroretinogram (ERG) recorded from electrodes implanted in the eye which was stimulated with flashes of monochromatic light. Three areas of maximum sensitivity were found by this method in the UV, blue/green and red. The relationship between the behavioural and electrophysiological sensitivity characteristics is explained with reference to visual systems of other Diptera which have previously been described.     

Changes in Odor Background Affect the Locomotory Response to Pheromone in Moths

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.     

Electroantennogram, wind tunnel and field responses of male Mediterranean corn borer, Sesamia nonagrioides, to several blends of its sex pheromone components

The electrophysiological and behavioural responses of male Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae) to several synthetic blends of its sex pheromone components were investigated. Electroantennogram (EAG) recordings showed the greatest response to a 77:8:10:5 blend of the four compounds found in the female glands: (Z)-11-hexadecen-1-yl acetate, (Z)-11-hexadecen-1-ol, (Z)-11-hexadecenal and dodecyl acetate. A logistic equation was fitted to the dose-response data and differences in relative EAG responses were analysed by deriving five parameters of this equation. Flight responses of males were also evaluated in a wind tunnel where the proposed blend (77:8:10:5) also elicited the best response. Field tests confirmed the laboratory findings as the 77:8:10:5 blend captured more S. nonagrioides males than other pheromone mixtures and showed more selectivity for male S. nonagrioides over other noctuid species, Discestra trifolii Rottenberg and Lacanobia oleracea L., but not for Mythimna unipuncta Haworth, a species with a sex pheromone composition almost identical to that of S. nonagrioides.     

Synthesis and biological activity of conformationally restricted gypsy moth pheromone mimics

The design and synthesis of a series of conformationally constrained mimics of gypsy moth sex pheromone, (+)-disparlure (7R,8S)-2-methyl-7,8-epoxyoctadecane, are described. The core structure of the mimics is derived from 5-(2′-hydroxyethyl)cyclopent-2-en-1-ol. Substituent optimization of the analogs was accomplished through the synthesis of mini-libraries and pure individual compounds, followed by electrophysiological experiments with male gypsy moth antennae. The electroantennogram results show that the analogs elicited weak to no antennal responses themselves. There was a clear structure–activity pattern for odorant activity, with ethyl substituents being best. Further, when puffed simultaneously with the pheromone, some of the compounds gave a significant enhancement of the antennal depolarization, indicating an additive or synergistic effect. A pure pheromone stimulus following a mixed compound/pheromone stimulus was generally not affected, with two exceptions: one compound enhanced and another inhibited a subsequent stimulus. The compounds also prolonged the stimulation of the antenna, which manifested itself in widened electroantennogram peaks. We tested the hypothesis that this prolonged stimulation may be due to the stabilization of a particular conformer of the pheromone-binding protein (PBP). Compounds that caused PBP2 to adopt a similar conformation than in the presence of pheromone also caused peak widening. This was not the case with PBP1.     

Behavioral and electrophysiological responses of Helicoverpa assulta, H. armigera (Lepidoptera: Noctuidae), their F1 hybrids and backcross progenies to sex pheromone component blends

Two sibling species, Helicoverpa assulta and Helicoverpa armigera both use (Z)-9-hexadecenal and (Z)-11-hexadecenal as their sex pheromone components but in almost reversed ratios, 93:7 and 3:97, respectively. H. assulta and H. armigera males performed upwind flight in response to the H. assulta sex pheromone blend (93:7). H. armigera responded strongly to the H. armigera blend (3:97), whereas H. assulta males remained inactive upon exposure to this blend. Both species gave clear dose-dependent electrophysiological responses to (Z)-11-hexadecenal. However, (Z)-9-hexadecenal evoked strong dose-dependent electrophysiological responses in H. assulta males but not in H. armigera. The two male F₁ hybrids exhibited similar behavioral responses to two sex pheromone blends and electrophysiological responses to two pheromone components as H. armigera males. This indicated that H. armigera genes appear dominant in determining the behavioral response and electrophysiological responses. Behavioral and electrophysiological responses of backcrosses of male F₁ hybrids (H. armigera female × H. assulta male) with female H. assulta and H. armigera were close to that of H. assulta and H. armigera, respectively. However, backcrosses of female F₁ hybrids (H. assulta female × H. armigera male) with male H. assulta and H. armigera showed reduced behavioral responses but normal electrophysiological responses compared to males of the respective parental line.     

Identification of the gland secreting oviposition-deterring pheromone in the cabbage seed weevil,Ceutorhynchus assimilis,and the mechanism of pheromone deposition

After laying an egg into a pod of Brassica napus, the female cabbage seed weevil, Ceutorhynchus assimilis, brushes the caudal setae of the eighth abdominal tergite (VIII UT) on the host pod as she walks along it, depositing oviposition-deterring pheromone (ODP). The VIII UT is periodically extended and withdrawn, thus repeatedly rubbing against the posterior fold of the seventh urotergite (VII UT) which bears the individual outlets of glandular epidermal cells. In post-diapause, sexually mature, gravid (i.e. oviferous) females (virgin or mated) the cells of this VII UT gland were hypertrophic, showing intense secretory activity. Extracts of VII UT from these individuals elicited strong electrophysiological responses from antennal club gustatory sensilla and deterred oviposition. In pre-diapause (sexually immature) females, the cells of the VII UT gland were neither hypertrophic nor active and an extract of their VII UT elicited no significant electrophysiological or behavioural response. Extract of female rectum was a less potent oviposition deterrent than VII UT extract and elicited an electrophysiological response similar to male rectum extract. An extract of ovarian calyces and ovaries elicited no behavioural response. We conclude that ODP is secreted by the epidermal cells of the VII UT posterior fold.     

Perception of potential sex pheromones and host-associated volatiles in the cotton plant bug,Adelphocoris fasciaticollis (Hemiptera: Miridae): morphology and electrophysiology

Highly developed chemoreception allows insects to detect foods, find mates, and escape natural enemies. We described the structures and distributions of antennal chemosensilla in Adelphocoris fasciaticollis Reuter by scanning electron microscopy and transmission electron microscopy. Seven major types of antennal sensilla were identified in adults of both sexes. Types 1 and 2 are sensilla chaetica and have thick cuticular walls with conspicuous grooves at their surfaces. Types 3 and 4 are multiporous sensilla trichodea with 1–3 dendrites located at the sensillum lymph, indicating a putative olfactory function. Types 5 and 6 are typical sensilla basiconica but share different characteristics in both external morphology and internal ultrastructure, and may be involved in the perception of host-associated odorants. The last sensilla were Böhm bristles. In addition to the morphological characterization, electrophysiological responses of antennal chemosensilla to 51 semiochemicals were investigated based on electroantennogram (EAG) recordings. Results revealed that different chemical stimuli elicited significantly different dose-dependent EAG responses, in which potential sex pheromone components and green leaf volatiles showed relatively higher EAG responses, but neither monoterpenes nor sesquiterpenes can elicit favorable EAG values. The results provided direct morphological and electrophysiological evidence that the adult antennae of A. fasciaticollis could function in searching for mates and host plants.     

Evidence of Chemical Signaling in the Sand Scorpion,Paruroctonus mesaensis (Scorpionida: Vaejovida)

This study presents evidence of intraspecific chemical communication in scorpions. The subject of our investigation was the nocturnal sand scorpion, Paruroctonus mesaensis. During the mating season, mature males show a sex-specific wandering behavior ostensibly directed at locating conspecific females that remain in the vicinity of their home burrows. Searching behavior was stimulated in the laboratory by releasing males onto substrates that had previously been occupied by females. Receptive males exhibited changes in locomotory behavior that favored occupation of the female-exposed area. Males occasionally displayed a precourtship behavior, called juddering, indicating the presence of a pheromone on the substrate. Juddering, and two newly described behavior patterns, tail-wagging and pedipalp-reaching, were also induced by solvent extracts of female cuticle. Most behavioral responses began vigorously within the first few s of stimulus contact and gradually adapted within 10 min. The potential importance of specialized sensory appendages, the pectines, for mediating chemosensitivity is discussed. These results are consistent with the hypothesis that mate identification and localization in sand scorpions are mediated in part by a contact sex pheromone.     

Patterns of response to sex pheromone by young and mature adult male cockroaches, Periplaneta americana

Male American cockroaches were isolated from females upon becoming adults and were exposed one single time to sex pheromone on days 1 to 11, 13, or 15 after adult ecdysis. The behavioural components of adult male sexual behaviour are rapid antennation, erect body posture, locomotion, running, wing raising, and abdominal extension. These components appear after the adult ecdysis in the same sequence as they appear in later adult life in response to different concentrations of sex pheromone. The appearance of these behavioural components is described using two theoretical concepts, threshold and a “sequencer”. It is proposed that the components are ordered sequentially before or during ecdysis by the same “sequencer” that organizes the responses to different concentrations of pheromone. The appearance of the components during development is due to decreasing thresholds as the adult male matures.     

Odor receptor responses of male gypsy and nun moths (Lepidoptera,Lymantriidae) to disparlure and its analogues

Summary The closely related species of the gypsy and the nun moth (Lymantria (Porthetria) dispar andL. monacha) were investigated with respect to their electrophysiological (electro-antennogram and single cell) responses to the sex pheromone (disparlure:cis-7,8-epoxy-2-methyl-octadecane) and 56 structurally related epoxides and the disparlure-precursor olefin. Within the limitations of reproducibility of the measurements the sequence of the effectiveness of all the tested compounds is nearly identical in both species, disparlure being every time the most effective compound. From this is deduced a high similarity in the receptor systems for female pheromones of the two moth species.     

Cyclic nucleotide-gated channel activation is not required for activity-dependent labeling of zebrafish olfactory receptor neurons by amino acids

The olfactory epithelium of fish is heterogeneous both with respect to the types of receptor cells (ORNs) present and the families of odorant receptors expressed in these cells. As a consequence of this diversity, the transduction cascade(s) activated by odorants has yet to be unambiguously established. In the current study, electrophysiological and activity-dependent labeling techniques were used to assess the role of the cyclic nucleotide-gated channel in zebrafish olfactory transduction. Both amino acid and bile salt odorants elicited robust electrophysiological responses, however, activity-dependent labeling of ORNs could be stimulated only by the amino acid odorants. An adenylate cyclase (AC) activator (forskolin) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) also elicited robust electrophysiological responses; generally larger than the responses elicited by either the amino acid or bile salt odorants. However, neither forskolin alone or a mixture of forskolin and IBMX stimulated activity-dependent labeling. Bathing the olfactory epithelium with forskolin, which presumably increased the intracellular concentration of cAMP, reduced the responses to bile salt odorants to a significantly greater extent than amino acid odorants. Collectively, these findings suggest that the transduction of amino acid input does not rely primarily on cyclic nucleotide-gated (CNG) channel activation and that CNG channel activation may be required for the transduction of bile salt input. Copyright Copyright 1999 S. Karger AG, Basel     

Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar)

Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.     

Pheromone receptor cells in the male moth Manduca sexta

Three types of pheromone receptor cells have been identified by electrophysiological recording from single antennal sensilla trichodea of the male sphinx moth Manduca sexta. These cells responded best to the pheromone components (E,Z)-10,12-hexadecadienal (type A receptor cell), (E,E,Z)-10,12,14-hexadecatrienal (type B), and (E,E,E)-10,12,14-hexadecatrienal (type C). Cell type B also responded to (E,Z)-11,13-pentadecadienal, which has been used experimentally as a pheromone substitute. In recordings from 20 trichoid hairs, 17 were found to be innervated by one cell of type A and one of type B; 3 trichoid hairs had cell types A and C.     

Resolution of pheromone pulses in receptor cells of Antheraea polyphemus at different temperatures

The ability of pheromone receptor cells of male Antheraea polyphemus (Saturniidae) to resolve stimulus pulses was determined at different temperatures (8°, 18°, 28°C). The cells were stimulated by repeated 20-ms puffs of the pheromone components (E, Z)-6, 11-hexadecadienyl acetate and (E, Z)-6,11-hexadecadienal. At higher temperatures, higher frequencies of stimulus pulses were resolved by the nerve-impulse response: about 1.25 pulses per second at 8°C, 2.5 pulses/s at 18°C and 5 pulses/s at 28°C. The decreased ability of receptor cells to resolve stimulus pulses at low temperatures may reduce the male moth's chance of reaching the pheromone source. The peak nerve-impulse frequency increased whereas the duration of nerve-impulse responses to single stimulus pulses decreased at higher temperatures. At a given temperature and stimulus intensity the peak nerveimpulse frequency decreased with shorter intervals between the stimulus pulses, but the duration of the responses remained almost constant. The time needed for recovery from adaptation caused by a single stimulus pulse was longer at lower temperatures. The aldehyde receptor cell recovered more quickly than the acetate cell. At low stimulus concentration, the resolution ability of the acetate cell was strongly decreased, whereas in the aldehyde cell it was only slightly impaired.     

Stimulus-response relationships of insect olfaction: Correlations among neurophysiological and behavioral measures of response

The inter-relationships among parameters of odorant concentration, the responses evoked in single olfactory receptor neurons, the whole-animal behavioral threshold, and the intensity of the behavioral response are examined for insect olfaction from the perspective of a new phenomenological model of the perceived intensity of single odorants. Electrophysiological and behavioral data compiled from diverse insect studies are analyzed in detail to determine the utility of the model for interpreting these studies and for designing new studies. The analysis of electrophysiological data indicates that the response of olfactory receptor neurons can be expressed as a power function of the odorant concentration. The behavioral thresholds to sex pheromone, predicted by the model from direct calculations using phenomenological parameters, are in good agreement with thresholds measured by bioassay. It is also shown that the interpretation of behavioral bioassay data in terms of concepts introduced by the model provides more information about the behavioral response than provided by the currently popular probit analysis. In addition, aspects of the physiological significance of power functions are discussed, and new methods are presented for distinguishing background activity from activity evoked in single olfactory neurons by an odor at threshold levels.