Electroantennogram and behavioural responses of the gypsy moth to enantiomers of disparlure and its trans analogues

The attractive power of both enantiomers of disparlure and its trans analog was tested electrophysiologically (electroantennogram = EAG) and behaviourally (whole organism response). EAG responses correlated very well with the behaviourally determined effectiveness sequence: cis(+)-disparlure was the most effective substance; the authentic racemic disparlure came second; cis(?)-disparlure inhibited the activity of cis(+)-disparlure; and trans enantiomers were not significantly different from the control. These results allow us to conclude that cis(+)-disparlure is the natural sex pheromone of the gypsy moth, and that the male antennae have a chiral receptor system in the antenna for the reception of the sex pheromone.     

Discrimination and production of disparlure enantiomers by the gypsy moth and the nun moth

ABSTRACT. Two odour receptor cells were physiologically identified within male antennal hair sensillae of the gypsy moth, Lymantria dispar L, and the nun moth, L. monacha L. In the gypsy moth, one cell responded to (+)-disparlure, while a neighbouring cell responded to (-)-disparlure. In the nun moth both cells responded to (+)-disparlure. The lack of sensitivity to (-)-disparlure in the nun moth was corroborated by electroantennogram (EAG) recordings, which indicated no affinity to this enantiomer. Single cell responses of male gypsy moth to different concentrations of the synthetic enantiomers of disparlure were then compared to responses elicited by hexane extracts of female glands of both species. The gypsy moth's extracts stimulated almost exclusively the receptor cell specialized for (+)-disparlure, while both cells were simultaneously stimulated by the extracts of the nun moths. From the response characteristic of the cells it is estimated that pheromone production of the nun moth is about 10% (+) and 90% (-)-disparlure, and that of the gypsy moth is almost 100% (+)-disparlure. Stimulation of the antenna of each species by female gland extracts of both species did not indicate the presence of receptors for other hexane elutable pheromone components in either species.     

Enantiomer and conformer recognition of (+) and (−)-disparlure and their analogs by the pheromone binding proteins of the gypsy moth,Lymantria dispar

Adult female gypsy moths produce a sex pheromone (+)-(7R,8S)-2-methyl-7,8-epoxyoctadecane, (+)-disparlure, to attract male gypsy moths. To better understand the recognition of (+)-disparlure by the male’s olfactory system, we synthesized racemic and enantiopure oxa and thia analogs of (+)-disparlure (ee > 98%). Ab initio calculations of the conformeric landscapes around the dihedral angles C_{5–6–7–8} and C_{7–8–9–10} of (+)-disparlure and corresponding dihedral angles of analogs revealed that introduction of the heteroatom changes the conformeric landscape around these important epitopes. The energy difference between HOMO and LUMO decreased after oxygen or sulfur was introduced into the backbone. Consistent with this, an enhancement of binding affinity between sulfur analogs and the pheromone-binding proteins (PBPs) was observed in vitro. Docking of the pheromone and analogs onto models of the two known PBPs of the gypsy moth revealed that the internal binding pocket of PBP1 showed higher selectivity than that of PBP2, consistent with in vitro binding assays. Further energy analysis revealed that enantiomers adopted different conformations with different energies when docked in the internal binding pocket of PBPs, resulting in enantiomer discrimination of PBPs towards disparlure and its analogs.     

Oxaspiropentane derivatives as effective sex pheromone analogues in the gypsy moth: electrophysiological and behavioral evidence

A number of oxaspiropentane derivatives (OXPs) were tested as potential (+)-disparlure analogues, with the aim of identifying any possible interaction of these compounds, be it additive, synergetic, or inhibitory, with the pheromone response in the male gypsy moth Lymantria dispar. As assessed by male electroantennograms, 2 OXPs, 2-decyl-1-oxaspiro[2.2]pentane (OXP-01) and 4-(1-oxaspiro[2.2]pent-2-yl)butan-1-ol (OXP-04), were found to be effective. OXP-01 had no stimulatory effect but strongly decreased the response to (+)-disparlure in a blend in a 1:1 ratio. By contrast, OXP-04 proved to be more stimulating than (+)-disparlure and also had an additive effect in the blend. Single-cell recordings from the sensilla trichoidea showed the activity of 2 cells, one of which responded to (+)-disparlure. OXP-01 reduced the stimulating effectiveness of pheromone by silencing the pheromone-responding unit when the 2 compounds were presented in blend, whereas OXP-04 mimicked the pheromone response, evidenced by exciting the pheromone-responding neuron when tested alone. Behavioral observations are in agreement with electrophysiological results.     

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.     

Application of lipase-catalyzed transformations for the synthesis of insect pheromones and related compounds

This review describes efficient means of preparing optically pure insect pheromones and related compounds via lipase-catalyzed enantioselective reaction on a large scale. (1) A new synthesis of the Japanese beetle pheromone, (R,Z)-(−)-5-(1-decenyl)oxacyclopentan-2-one, established by a combination of two lipase-catalyzed transformation was demonstrated. (2) A chemico-enzymatic procedure for the syntheses of both enantiomers of cupreous chafer beetle pheromone, (R,Z)- and (S,Z)-5-(1-octenyl)oxacyclopentan-2-one, was described. (3) An optical resolution of (±)-2,3-epoxy-8-methyl-1-nonanol, the key intermediate of the synthesis of gypsy moth pheromone, was demonstrated. (4) A practical chemico-enzymatic synthesis of (+)-disparlure in large scale was demonstrated. (5) A facile synthesis of carboxyalkyl acrylate, which is special monomers in the synthesis of the new polymers, by two lipase-catalyzed regioselective reactions was described.     

Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar

The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane, (+)-disparlure, as a sex pheromone. The (-) enantiomer of the pheromone is a strong behavioral antagonist. Specialized sensory hairs, sensillae, on the antennae of male moths detect the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) transports the odorant to the sensory neuron. We have expressed the two PBPs found in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study pheromone binding under equilibrium conditions, we developed and validated a binding assay. We have addressed the two major problems with hydrophobic ligands in aqueous solution: (1) concentration-dependent adsorption of the ligand on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate for the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 has a higher affinity for the (-) enantiomer, while PBP2 has a higher affinity for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polyphemus (Apol-3) bound the disparlure enantiomers more weakly than either of the L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers. We have observed extensive aggregation of both L. dispar PBPs and an increase in pheromone binding at high (>2 microM) PBP concentrations. We present a model of disparlure binding to the two PBPs.     

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.     

Pheromone-mediated optomotor anemotaxis and altitude control exhibited by male oriental fruit moths in the field

Abstract. In the field over short grass, pheromone-stimulated oriental fruit moth males, Grapholita molesta (Busck), flying under high windspeeds tended to steer courses more into the wind and to increase their airspeeds compared with those flying in low windspeeds.Thus, optomotor anemotaxis enabled the males to steer relatively consistent upwind track angles and to maintain an upwind progress of between c. 50–100 cm/s despite variable wind velocities.Zigzagging flight tracks were observed at both 10 m and 3 m from the source, as were tracks with no apparent zigzags.Transitions from casting to upwind flight or vice-versa were observed.The durations of the intervals between reversals during both upwind zigzagging flight and casting were consistent with those observed in previous wind-tunnel experiments.The control of altitude was more precise during upwind zigzagging flight than during casting.In general, the side-to-side deviations in the tracks were greater than the up-and-down deviations, with both the side-to-side and vertical distances and their ratios being consistent with previous wind-tunnel studies of pheromone-mediated flight.One difference between the field and laboratory flight tracks was that males in the field exhibited much higher airspeeds than in the wind tunnel.Males occasionally were observed to progress downwind faster than the wind itself, and further analysis showed that they were steering a downwind course in pheromone-free air following exposure to pheromone, which is the first time this has been recorded in moths.We propose that such downwind flight may aid in the relocation of a pheromone plume that has been lost due to a wind-shift, by enabling the moth to catch up to the pheromone as it recedes straight downwind away from the source.     

Impact of (Z)-7-dodecenol and turbulence on pheromone-mediated flight manoeuvres of male Trichoplusia ni

Abstract. Turbulence and chemical noise are two factors which may influence pheromone-mediated flight manoeuvres of a moth in natural habitats. In this study, the effects of turbulence and the behavioural antagonist (Z)-7-dodecenol on flight manoeuvres of male Trichoplusia ni (Hübner) were evaluated in a wind tunnel. Male moths increase airspeed and course angles when turbulence is increased. This leads to significant increases in the length of flight tracks, but significant reductions in the time taken to reach a pheromone source. In less disturbed pheromone plumes, distributions of course angles and track angles of male T.ni show a prominent peak centred about 0° relative to the upwind direction, indicating that moths can temporarily steer directly upwind toward a pheromone source.
When (Z)-7-dodecenol is released 10 cm upwind of a pheromone source to form an overlapping plume downwind, course angles, airspeeds and ground-speeds of male T.ni are reduced significantly compared with those in uncon-taminated pheromone plumes. This results in a longer flight time to reach a pheromone source. The decrease in flight speed would decrease the rate of contact with filaments, and thereby perhaps allow the moth to detect uncon-taminated pheromone filaments independently from filaments containing the behavioural antagonist.     

Height control by free-flying Drosophila

ABSTRACT. In a horizontal wind tunnel, Drosophila flew at almost constant height along tracks up to 2 m long. The flies rose or sank only slowly when it was so dark that they no longer responded to movements of the tunnel floor, suggesting that their height control is mediated, at least partly, by responses to their movement relative to the air. In the light, the flies maintained height better than in the dark and were very responsive to movements around them. They faithfully followed the up and down movements of horizon screens at their sides whether they were flying in still air or against a wind, even in the presence of many other stationary visual cues. The flies did not respond by compensatory height changes to real vertical movements of a patterned horizontal disc beneath them, nor to changes in the size of the floor pattern. They did respond to horizontal acceleration of the floor pattern in the direction opposite to their flight (optically simulating a descent by the fly), by an apparently compensatory increase in height, but they also rose (instead of sinking) in response to floor acceleration in the direction of their flight. When the floor was accelerated in either direction they showed compensatory groundspeed-controlling responses. The increases in height might be alarm responses to sudden movements in the visual field beneath them. Both speed and height changing responses to floor movement were reduced when the number of stationary visual cues was increased. Drosophila thus control their height mainly by responses to the apparent movement of nearby visual cues at round about their own height.     

Anemotactic orientation of gypsy moth males and its modification by the attractant pheromone (+)-disparlure during walking

ABSTRACT. Tracks of dewinged gypsy moth males, Lymantria dispar L. (Lymantriidae), walking upwind in an airstream without pheromone consist of marked alternations between more or less straight upwind segments, partly with an arcadic structure, and twisted segments. This apparently complicated behaviour can, however, simply be explained by a superposition of noise and two turning commands: an upwind turning tendency, derived from the anemoreceptive system, which represents an average of the moth's angular positions over a period of time; and an internal turning tendency which consists of strong but brief bursts. These bursts are produced intermittently; successive bursts do not necessarily alternate polarity. Amputation of one antenna increases the probability of bursts towards the amputated side; therefore a separate burst source is postulated for each antenna. In the presence of the attractant pheromone (+)-disparlure, the anemotactic signal is weighted higher; twisted segments are, therefore, less pronounced.
There is a chemotropotactical component involved in the male's orientation. The tropotactical signal, dependent on the difference of odour concentration perceived by the left and right antenna, competes with the upwind turning tendency.     

Towards pheromone-based monitoring of nun moth, Lymantria monacha (L.) (Lep., Lymantriidae) populations

The research objective was to develop pheromone-based monitoring of the nun moth, Lymantria monacha (L.), an important defoliator of spruce and pine forests in central Europe. In 38 spruce or pine forests in central Europe, captures of male L. monacha in nonsaturating Unitraps and saturating Delta sticky traps baited with 0.2, 2, 20, or 200 μg of the L. monacha (pheromone) volatile blend [(±)-disparlure, (±)-monachalure, and 2-methyl- Z 7-octadecene at a 20 : 20 : 1 ratio] were compared with estimates of population densities obtained by counts of larval faecal pellets, pupal cases, and adult moths resting on tree trunks. Total captures of male L. monacha throughout the flight season in both types of trap were correlated with numbers of larval faecal pellets, irrespective of pheromone dose. Nonsaturating Unitraps baited with 2 μg of the L. monacha volatile blend seem to provide a cost-effective tool for monitoring densities of L. monacha populations. Long-term testing of this monitoring system has been initiated to substantiate the quantitative relationship between larval populations and trap captures of male L. monacha and to determine the threshold number of captured male moths that indicates an incipient outbreak.     

Cowpea weevil flights to a point source of female sex pheromone: analyses of flight tracks at three wind speeds

Abstract.  Two-day-old male cowpea weevils, Callosobruchus maculatus, fly upwind to a point source of female sex pheromone at three wind speeds. All beetles initiating flight along the pheromone plume make contact with the pheromone source. Analysis of digitized flight tracks indicates that C. maculatus males respond similarly to moths tested at several wind speeds. Beetles' mean net upwind speeds and speeds along their track are similar ( P  > 0.05) across wind speeds, whereas airspeeds increase ( P <  0.01) with increasing wind speed. Beetles adjust their course angles to fly more directly upwind in higher wind speeds, whereas track angles are almost identical at each wind speed. The zigzag flight paths are generally narrow compared with most moth flight tracks and interturn distances are similar ( P  > 0.05) at the wind speeds employed. The frequency of these counterturns across the wind line is almost constant regardless of wind speed, and there is little variation between individuals. The upwind flight tracks are more directly upwind than those typically seen for male moths flying upwind toward sex pheromone sources. Male moths typically produce a bimodal distribution of track angles to the left and right of the windline, whereas C. maculatus males' track angles are centred about 0°. Preliminary examination of two other beetle species indicates that they fly upwind in a similar fashion.     

An analysis of anemotactic zigzagging flight in male moths stimulated by pheromone

ABSTRACT. The zigzagging behaviour of male Plodia interpunctella flying up a plume of sex pheromone was investigated in a horizontal wind tunnel by detailed analysis of the moths' ground tracks, groundspeeds, orientations and airspeeds. The moths ‘homed in’ on the source of the pheromone plume by progressively reducing airspeed and turning more into wind, thereby reducing groundspeed and the distance between track reversals and so narrowing down their zigzags (Fig. 16). Track angles and times between reversals were unaffected. Removing the wind-borne pheromone plume while a moth was flying along it confirmed that zigzagging can be an anemotactic response to losing the scent rather than a chemotactic response to the plume. For the first 1–2 s after the moth entered pheromone-free air the zigzagging was indistinguishable from that shown when the plume remained; thereafter it widened progressively until the moths were flying to and fro at c. 90° to the wind. The after-effect of odour stimulation persisted for many zigs and zags and many seconds (Figs. 4 and 5). Moths flying along pheromone plumes compensated efficiently for differences of wind speed, showing similar distributions of track angles to wind, and of ground-speeds, in winds of 0.1, 0.2 and 0.3 ms^-1 (Figs. 12 and 13). Groundspeed varied with track angle to wind and this relationship was also similar in the three wind speeds (Fig. 14). This constancy of track angles and groundspeeds was due to the moths both increasing their airspeeds and turning more into wind at the higher wind speeds (Fig. 17). Thus the direction of the apparent movement of the ground pattern beneath the moths varied with wind speed. It is inferred that the moths, although unable to sense the wind directly, are able to compensate for changes in wind speed by integrating the wind-dependent optomotor input with information about their own airspeed, or with information about their own turning movements. Maintaining some ‘preferred’ relationship between these inputs by adjustments of orientation and airspeed, would then serve to maintain a given combination of track angle and groundspeed independently of wind speed. The preferred relationship is repeatedly re-set by the changing olfactory input from the pheromone plume, which also controls the switching between left and right of the upwind direction.     

Evidence of active or passive downwind dispersal in mark–release–recapture of moths

Modelling moth dispersal in relation to wind direction and strength could greatly enhance the role of pheromone traps in biosecurity and pest management applications. Anemotaxis theory, which describes moth behaviour in the presence of a pheromone plume and is used as a framework for such models. Currently, however, that theory includes only three components: upwind, zigzagging, and sideways casting behaviour. We test anemotaxis theory by analysing the data from a series of mark–release–recapture experiments where the wind direction was known and the insects were trapped using an irregular grid of pheromone traps. The trapping results provide evidence of a downwind component to the flight patterns of the released insects. This active or passive downwind dispersal is likely to be an appetitive behaviour, occurring prior to the elicitation of pheromone‐oriented flight patterns (pheromone anemotaxis). Given the potential for significant displacement during downwind dispersal, this component will have impact on final trap captures and should be considered when constructing moth dispersal models.     

Instrumental measurement of the gypsy moth pre-flight behavioural response to pheromone

ABSTRACT. Vibrations of the thorax and electrical activity (EMG) of gypsy moth flight muscles were recorded during wing fanning following pheromone stimulation. The percentage of positive responses and durations of bursts of flight muscle activity increased with the logarithm of pheromone dose, whereas latency decreased. The results correlated well with wing fanning responses of freely moving gypsy moths exposed to nearly identical stimulus conditions. Typical dose-response curves in the range of 0.04-400ng disparlure were obtained in both types of experiments. These methods provide an electrical analogue of wing fanning behaviour.     

Comparison of psychic emergence reactions after (+/-)-ketamine and (+)-ketamine in mice

Ketamine is a racemic mixture containing equal parts of (+)-ketamine and (-)-ketamine. The ketamine enantiomorphs are different in anesthesia and psychic emergence reactions after anesthesia. Therefore, (+)-ketamine was compared with racemic ketamine in a number of randomized studies in volunteers and patients. However, their relations remain controversial. In the present studies, the psychic emergence reactions after injection of (+/-)-ketamine and (+)-ketamine were compared in mice. At equimolar doses, the (+)-isomers elicited episodes of hypnosis nearly 1.4-fold more potent than those of the racemic ketamine. After the administration of equihypnotic doses of (+)-ketamine and (+/-)-ketamine, the posthypnotic stimulation of locomotor activity, stereotype behavior and 5-HT-induced head-twitch response by the (+)-enantiomorph was significantly less intense than that of racemic ketamine. In receptor binding test, (+)-ketamine showed a higher affinity for NMDA receptor than that of (+/-)-ketamine, while (+)-ketamine and (+/-)-ketamine showed no affinity for dopamine D2 and serotonin 5-HT2 receptor. These results suggest that the (+)-ketamine has fewer posthypnotic side effects than (+/-)-ketamine when (+)-ketamine and (+/-)-ketamine were administered at equihypnotic dosages and that dopamine D2 and serotonin 5-HT2 receptor were not involved in the effects of (+)-ketamine and (+/-)-ketamine.     

Sulcatol: population aggregation pheromone in the scolytid beetle, Gnathotrichus sulcatus

The population aggregation pheromone produced by males of Gnathotrichus sulcatus, a timber pest, has been identified from boring dust as a $\text{65}\text{35}$ mixture of the (S)-(+) and the (R)-(?) enantiomers of 6-methyl-5-hepten-2-ol. In field studies beetles were attracted in a 2·65 female: 1 male ratio by racemic synthetic pheromone.     

Flight-phase and visual-field related optomotor yaw responses in gregarious desert locusts during tethered flight

Tethered flying desert locusts, Schistocerca gregaria, generate yaw-torque in response to rotation of a radial grating located beneath them. By screening parts of the pattern, rotation of the unscreened grating turned out to induce a compensatory steering (by pattern motion within transversally oriented 90° wide sectors) as well as an upwind/downwind turning response (by pattern motion within the anterior ventral 90° wide sector). The strength and polarity of responses upon the unscreened grating results from a linear superposition of these two response components. The results are discussed with regard to a functional specialization of eye regions.In a typical experiment, 3 consecutive flight-phases, assumed to mirror start, long-range flight, and landing of a free-flying locust, were distinguished. They may result from a time dependent variation of the polarity and relative strength of upwind/downwind turning and compensatory steering responses. Starting and landing phases were under strong optomotor control and were dominated by the high-gain compensatory steering. In contrast, the phase of long-range flight was under weak optomotor control resulting from a low gain in both of the two response components. The biological significance of this variable strength of optomotor control on free flight orientation of swarming locusts is discussed.