Changes in Odour Intermittency Influence the Success and Search Behaviour During Orientation in the Crayfish (Orconectes Rusticus)

Aquatic organisms may use different mechanisms to locate an odour source, including odour-gated rheotaxis and chemotaxis. However, the effects of spatial and temporal information contained within odour signals in guiding orientation are under debate. In nature, turbulence and plume meandering give rise to signal intermittency, which organisms must overcome to locate an odour source successfully. In this study, crayfish were presented with odour plumes formed by a continuously released jet or a pulsed jet ranging from 0.5 to 3 Hz. Crayfish oriented significantly differently in pulsed odour plumes differed significantly from than in a continuously released jet plume. In a continuous odour plumes, crayfish walked faster, had more accurate heading and turning angles, and stopped less often. The results suggest that crayfish are able to orient in a pulsed odour plumes, but that the spatial and temporal complexity of a continuous released odour plume allowed them to be more successful to find the odour source.     

Fine-scale structure of odour plumes in relation to insect orientation to distant pheromone and other attractant sources

ABSTRACT. This paper describes an experimental method for simulating an odour plume in the field so that its fine-scale characteristics may be determined. It was found that the 'odour' arrived at a series of fixed points up to 15 m from the source in a series of discrete bursts, which were widely distributed in time but were typically 0.1 s long and 0.5 s apart. The strengths of the bursts were also found to be widely distributed and some contained considerable fluctuations. Thus an odour plume is not continuous, but intermittent, and appears at a fixed point downwind of the odour source as a series of bursts of odour which are variable in strength and duration. The distribution of the length of bursts and the time between them does not greatly vary with distance from source. Although concentration does decay with distance from source, instantaneous measurement would not be a reliable guide to the mean concentration and hence to position relative to source. To obtain reliable positional information from the odour plume an insect would have to average received stimuli over many seconds. More information is therefore required about the dynamics of insects' olfactory responses.     

Field and laboratory electroantennographic measurements of pheromone plume structure correlated with oriental fruit moth behaviour

Abstract. Peak-to-trough electroantennogram amplitudes (bursts), caused by the individual filaments of a plume of female pheromone, diminish as high-emission-rate sources are approached by male Grapholita molesta , and this reduction is correlated with in-flight arrestment (ceasing to advance upwind). These findings are consistent with the hypothesis that one cause of in-flight arrestment in response to high-concentration point sources is the attenuation of the peak-to- trough amplitudes close to the source. High burst frequency, high pheromone flux, or low levels of continuous neuronal activity all are less well correlated with arrestment. Rather, arrestment appears due to a reduction of chemosensory input to the CNS during flight up the plume, even though the actual molecular concentration continues to increase. In a laboratory wind tunnel, upwind flight initiation by more than 20% of males was elicited only by pheromone source concentrations evoking significant fluctuations in EAG amplitudes at downwind release points. The burst frequencies that evoked high levels of upwind flight initiation ranged from a mean of 0.4-2.2 bursts/s. Because a previous study revealed that flying male G. molesta change their course angle within 0.15 s of losing or contacting pheromone, these EAG burst frequencies indicate that during flight in a pheromone plume, many manoeuvres are probably made in response to contact with individual plume filaments. Thus, upwind flight tracks may be shaped by hundreds of steering reactions in response to encounters with individual pheromone filaments and pockets of clean air. Field-recorded EAGs reveal that burst amplitudes diminish from 3 to 30 m downwind of the source, whereas burst frequencies do not, averaging c. 1/s at 3, 10 and 30 m downwind.     

Effects of pheromone plume structure and visual stimuli on the pheromone-modulated upwind flight of male gypsy moths (Lymantria dispar) in a Forest (Lepidoptera: Lymantriidae)

The pheromone-modulated upwind flight ofLymantria dispar males responding to different pheromone plume structures and visual stimuli designed to mimic trees was video recorded in a forest. Males flying upwind along pheromone plumes of similar structure generated tracks that were similar in appearance and quantitatively similar in almost all parameters measured, regardless of the experimentally manipulated visual stimuli associated with the pheromone source. Net velocities, ground speeds, and airspeeds of males flying in point-source plumes were slower than those of males flying in the wider, more diffuse plumes issuing from a cylindrical baffle. The mean track angle of males flying in plumes issuing from a point source was greater (oriented more across the wind) than that of males flying in plumes issuing from a transparent cylindrical baffle. Males flying in point-source plumes also turned more frequently and had narrower tracks overall than males responding to plumes from a cylindrical baffle. These data suggest thatL. dispar males orienting to pheromone sources (i.e., calling females) associated with visible vertical cylinders (i.e., trees) use predominantly olfactory cues to locate the source and that the structure of the pheromone plume markedly affects the flight orientation and the resultant track.     

Close encounters: contributions of carbon dioxide and human skin odour to finding and landing on a host in Aedes aegypti

In a wind‐tunnel study, the upwind flight and source location of female Aedes aegypti to plumes of carbon dioxide (CO₂) gas and odour from human feet is tested. Both odour sources are presented singly and in combination. Flight upwind along the plumes is evident for both CO₂ and odour from human feet when the odours are presented alone. Similarly, both odour sources are located by more than 70% of mosquitoes in less than 3 min. When both CO₂ and odour from human feet are presented simultaneously in two different choice tests (with plumes superimposed or with plumes separated), there is no evidence that females orientate along the plume of CO₂ and only a few mosquitoes locate its source. Rather, the foot odour plume is navigated and the source of foot odour is located by over 80% of female Ae. aegypti. When a female is presented a plume of CO₂ within a broad plume of human foot odour of relatively low concentration, the source of CO₂ is not located; instead, flight is upwind in the diffuse plume of foot odour. Although upwind flight by Ae. aegypti at long range is presumably induced by CO₂ and the threshold of response to skin odours is lowered, our findings suggest that, once females have arrived near a prospective human host, upwind orientation and landing are largely governed by the suite of human odours, whereas orientation is no longer influenced by CO₂.     

Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide

Electroantennogram (EAG) measurements were recorded from the antennae of male and female codling moth, Cydia pomonella L., to determine whether adult moths exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide experience a decline in their antennal reception and thus olfactory sensitivity. Such a phenomenon would offer a possible mechanism for the previously reported decreased responsiveness from moths treated with methoxyfenozide to pheromone‐ and plant volatile‐based monitoring lures. Mean EAG data revealed that the antennae from methoxyfenozide‐treated male moths appear to be just as sensitive to various doses of synthetic codlemone as the antennae from the control and surfactant‐treated moths, but they appeared to be less sensitive to the pheromone component 12OH (collected from female effluvia) than the control male antennae. Mean male EAG responses to the pheromone components E8,E10‐12Al and codlemone collected from methoxyfenozide‐treated females were significantly less than the responses towards those two pheromone components collected from the control and surfactant‐treated females. Female moth exposure to methoxyfenozide did not negatively impact the sensitivity of female antennae to the plant volatile pear ester, but it did towards the apple volatile butyl hexanoate. Data from this study show that adult C. pomonella exposure to methoxyfenozide‐treated surfaces does appear to negatively impact, in a minor way, the (i) olfactory sensitivity (or detection) of male antennae towards some components of the female sex pheromone, (ii) the female antennal sensitivity towards a key apple volatile and (iii) the attractiveness of female pheromone effluvia.     

Ultrastructure and potential role of integumentary glandular cells in adult male and female Callosobruchus subinnotatus (Pic) and C. maculatus (Fabricius) (Coleoptera : Bruchidae)

Transmission and scanning electron microscopy methods were used to study the ultrastructure of integumentary glandular cells that may be involved in the production of sex pheromones and other semiochemicals in Callosobruchus subinnotatus and C. maculatus (Coleoptera : Bruchidae). Additionally, we measured electroantennogram (EAG) responses of male and female antennae to solvent extracts and glassadsorbed volatiles from both sexes of C. maculatus in order to localize the source of the putative sex pheromone. Both species have numerous cuticular pores on the abdomen and thorax. These pores open via an epicuticular duct into a single type 3 secretory cell. Solvent extracts of the pygidium from the female elicit the highest EAG responses from male antennae, suggesting this area of the abdomen to be the source of the putative sex pheromone in C. maculatus.     

Multiphasic On/Off Pheromone Signalling in Moths as Neural Correlates of a Search Strategy

Insects and robots searching for odour sources in turbulent plumes face the same problem: the random nature of mixing causes fluctuations and intermittency in perception. Pheromone-tracking male moths appear to deal with discontinuous flows of information by surging upwind, upon sensing a pheromone patch, and casting crosswind, upon losing the plume. Using a combination of neurophysiological recordings, computational modelling and experiments with a cyborg, we propose a neuronal mechanism that promotes a behavioural switch between surge and casting. We show how multiphasic On/Off pheromone-sensitive neurons may guide action selection based on signalling presence or loss of the pheromone. A Hodgkin-Huxley-type neuron model with a small-conductance calcium-activated potassium (SK) channel reproduces physiological On/Off responses. Using this model as a command neuron and the antennae of tethered moths as pheromone sensors, we demonstrate the efficiency of multiphasic patterning in driving a robotic searcher toward the source. Taken together, our results suggest that multiphasic On/Off responses may mediate olfactory navigation and that SK channels may account for these responses.     

A portable electroantennogram sensor for routine measurements of pheromone concentrations in greenhouses

Mating disruption using pheromone dispensers is an emerging insect control method in greenhouses in the Netherlands. For routine measurements of the pheromone level in greenhouses a portable and compact device based on electroantennography (EAG) was developed. The instrument was tested in different greenhouses under varying conditions using antennae of males of the noctuid moth Chrysodeixis chalcites. Relative differences in pheromone concentration between greenhouses loaded with pheromone dispensers and control greenhouses could be measured in a reliable way. Changes in pheromone concentration were clearly demonstrated by measurements during the growing season. The advantages of the instrument presented over previously described devices are its true portableness, ease of use and uncomplicated measuring principle. The device can be operated routinely by non-experts, and many measurements can be made in a short period of time. Preliminary outdoor measurements using antennae of male Cydia pomonella as sensor showed the potential of the instrument for use in the open field. The portable EAG sensor described here may provide a practical means for monitoring pheromone concentration and dispersal from dispensers applied for mating disruption.     

How insects sense olfactory patches – the spatial scaling of olfactory information

When searching for resources in heterogeneous environments, animals must rely on their abilities to detect the resources via their sensory systems. However, variation in the strength of the sensory cue may be mediated by the physical size of the resource patch. Patch detection of insects are often predicted by the scaling of sensory cues to patch size, where visual cues has been proposed to scale proportional to the diameter of the patch. The scaling properties of olfactory cues are, however, virtually unknown. Here, we investigated scaling rules for olfactory information in a gradient of numbers of odour sources, relevant to odour‐mediated attraction under field conditions. We recorded moth antennal responses to sex pheromones downwind from pheromone patches and estimated the slope in the scaling relationship between the effective length of the odour plumes and the number of odour sources. These measurements showed that the effective plume length increased proportional to the square root of the number of odour sources. The scaling relationship, as estimated in the field experiment, was then evaluated against field data of the slope in the relationship between trap catch and release rate of chemical attractants for a wide range of insects. This meta‐analysis revealed an average slope largely consistent with the estimated scaling relationship between the effective plume length and the number of odour sources. This study is the first to estimate the scaling properties of olfactory cues empirically and has implications for understanding and predicting the spatial distributions of insects searching by means of olfactory cues in heterogeneous environments.     

Quantitative measurement of general odorant using electroantennogram of male silkworm moth,Bombyx mori

The investigation of electroantennogram (EAG) using insect antennae has been primarily focused on the measurement of insect pheromone. Insect has highly specialized olfactory receptors inside their antennae. In this paper, EAG was applied to detect general odorants and the feasibility of this system for the olfactory biosensor was investigated. Electroantennogram measurement was carried out using the antennae of male silkworm moth,Bombyx mori, and ammonia gas as the model odorant. EAG parameters including peak amplitude, decay, and level were analyzed for the quantitative measurement. The peak amplitude increased linearly with the ammonia concentration and the reproducible electrical signals were generated at least for 2 hrs after the antenna was cut off from the silkworm moth.     

Factors affecting the antennal and behavioural responses of the saw-toothed grain beetle Oryzaephilus surinamensis to food odour and aggregation pheromone

ABSTRACT. The effects of insect age, sex, mated state and culture density on the attraction of adult saw-toothed grain beetles, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae), to sources of food odour and synthetic aggregation pheromone were investigated. In single-insect behavioural assays, insect age affected the attraction to the food odour, but not to the synthetic aggregation pheromone. Conversely, there were sexual differences in the attraction to the aggregation pheromone, but not to the food odour. The effects of the same factors on the antennal response to food odour and synthetic pheromone were also investigated, using the electroantennogram (EAG) technique. Insect age affected the EAG response to food odour, but not to the synthetic pheromone. There were no sexual differences in the EAG responses to either food odour or synthetic pheromone.
The correlation of factors affecting both behavioural and antennal responses, and the possible role of changes in peripheral olfactory receptor sensitivity in the modulation of behaviour are discussed.     

Real-time measurement of pheromone release from individual female moths and synthetic dispensers in a wind tunnel by recording of single receptor-neurone responses

Measurements of single neurone activity in the peripheral pheromone receptors of male Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) were performed in a wind tunnel using a portable electrophysiological recording unit. Filter paper and rubber septa loaded with synthetic sex pheromone, as well as individual conspecific female glands, were used as pheromone sources. Recordings, up to 3 h long, were analysed for temporal variation in spiking activity. The recordings were performed 2 m downwind of the source, where the pheromone plume had a width of approximately 12 cm, as could be measured with the single cell preparations. The system allowed reliable measurements of relative pheromone concentration with a 20-s time resolution. The release rate from rubber septa loaded with pheromone was more or less constant over time, whereas the release rate from filter paper loaded with pheromone decreased to one tenth of the initial value within 6 min from the application of the pheromone. The release of pheromone from female pheromone glands was pulsed with an interval of 2–10 min between bursts. This pulsing was not caused by retraction of the gland, as the glands were forcibly extruded during the entire experiment, but should reflect variation in transport of pheromone to the gland surface and subsequent release. The demonstrated stability of the preparations using tungsten electrodes, the reliable monitoring of female-produced pheromone plumes at several metres distance, and the time resolution obtained are important steps towards field monitoring of natural pheromone plumes, as well as pheromone concentration and distribution in applications for mating disruption.     

Helicoverpa zea males (Lepidoptera: Noctuidae) respond to the intermittent fine structure of their sex pheromone plume and an antagonist in a flight tunnel

Abstract. We investigated the behavioural response of male Helicoverpa zea (Boddie) to the fine-scale structure of an odour plume experimentally modified in a wind tunnel by using an air-pulsing device. Male H. zea flew upwind to pulsed filaments of a binary pheromone blend of (Z)-11-hexadecanal (Z11-16:Ald) and (Z)-9-hexadecanal (Z9-16:Ald) in the ratio of 20:1. Sustained upwind flight in experimentally altered intermittent plumes was dependent on concentration, as well as the frequency of generation of odour filaments. At a loading of 10μg of the major pheromone component, Zll-16:Ald, which gave an emission rate of approximately that released by a female H. zea , sustained upwind flight and source contact correlated positively with filament delivery rate, becoming significant at a minimum filament delivery rate of 2/s. Decreases in upwind progress and source location were recorded at a loading of 1 μg of Z11-16:Ald. At this suboptimal dosage, a high filament generation rate of 10/s was necessary for significant upwind progress and source contact. When an interspecific compound: (Z)-11-hexadecenyl acetate (Z11-16:OAc), was added to the attractive pheromone binary aldehyde blend of H. zea at a proportion of 10% of the major pheromone component, and pulsed from the same source, there was a significant reduction in sustained upwind progress and source location by males, indicating that Z11-16:OAc is antagonistic to the upwind progress of H. zea. However, Z11-16:OAc was less antagonistic when its filaments were isolated and alternated with pheromone filaments, indicating a strong effect of the synchronous arrival of odour filaments on the antenna needed for antagonism of upwind flight.     

Flight behaviour of Cadra cautella males in rapidly pulsed pheromone plumes

Abstract Airborne pheromone plumes in wind comprise filaments of odour interspersed with gaps of clean air. When flying moths intercept a filament, they have a tendency to surge upwind momentarily, and then fly crosswind until another filament is intercepted. Thus, the moment-to-moment contact with pheromone mediates the shape of a flight track along the plume. Within some range of favourable interception rates, flight tracks become straighter and are headed more due upwind. However, as the rate of interception increases, there comes a point at which the moth should not be able to discern discreet filaments but, rather, should perceive a 'fused signal'. At the extreme, homogeneous clouds of pheromone inhibit upwind progress by representative tortricids. In a wind tunnel, Cadra cautella (Walker) (Lepidoptera: Pyralidae) were presented with 10 ms pulses of pheromone at a repetition rate of 5, 10, 17 and 25/s and a continuous, internally turbulent plume. Pulse size and concentrations were verified with a miniature photoionization detector sampling surrogate odour, propylene, at 100 Hz. Male moths maintain upwind progress even at plumes of 25 filaments/s. Furthermore, moths exhibited greater velocities and headings more due upwind at 17 and 25 Hz than at the lower frequencies or with the continuous plume. It is hypothesized that either C. cautella possesses a versatile sensory system that allows the resolution of these rapidly pulsed pheromone plumes, or that this species does not require a 'flickering' signal to fly upwind.     

Effect of the fine-scale structure of pheromone plumes: pulse frequency modulates activation and upwind flight of almond moth males

Abstract. The effects of plume intermittency and volume on behavioural and flight responses to pheromone of male Cadra cautella (Walker) (Lepidoptera: Phycitinae) were investigated in a pulling wind tunnel. The fine-scale structure of turbulent pheromone plumes was mimicked and manipulated using a pulser device that generated continuous ribbon plumes or intermittent plumes with defined pulse frequency and volume. As pulse frequency increased from 0.6 to 5 Hz and injected volume increased from 0.5 to 5 mls^-1, males flew progressively higher air and ground speeds, turned less frequently, and steered smaller course angles, resulting in straighter flight tracks. The faster the frequency of pulses and the greater the volume of the plume, the higher the proportion of males responding, the shorter their latencies, and the less time spent in the behaviour. Flight tracks of male C.cautella to point sources of pheromone depend on the frequency of filaments encountered.     

Influence of plume structure and pheromone concentration on upwind flight of Cadra cautella males

Abstract. The effects of pheromone plume structure and its concentration on the pheromone-mediated flight of male Cadra cautella (Lepidoptera: Phycitinae) were investigated in a laminar-flow wind tunnel. When two C. caurella males flew simultaneously along a ribbon plume of mixed smoke and pheromone, their inflight behaviour was dependent on the instantaneous structure of the plume they encountered. When a male intercepted an intact ribbon filament, he sustained a crosswind course, whereas when he intercepted a turbulent filament (created by an upwind male fragmenting the ribbon plume), he adopted a flight course more due upwind. These results indicate that C. cautella males altered their in-flight manoeuvres in response to instantaneous changes in the fine structure of the pheromone plume. We also demonstrated that differences in the fine structure of the plume had more influence on the flight pattern of C. cautella males than a 1000-fold range in pheromone dose. The size of the plume was increased by adding wind deflectors upwind of the pheromone source, independent of source dosage, males following ribbon plumes flew slow zigzag tracks, whereas males following large, turbulent plumes flew directly to the source in fast, straight tracks with less counterturning.     

Flight of male Cadra cautella along plumes of air and pheromone superimposed on backgrounds of pheromone

Male Cadra cautella were presented with five heterogeneous pheromone clouds (created from source doses of 0, 0.01, 1, 100, and 10 000 ng) with and without superimposed plumes of either clean air or sex pheromone in a wind tunnel. Moths provided with the lowest doses of background clouds without a superimposed plume did not fly upwind. Moths provided with higher doses of background clouds, with or without superimposed air plumes, increased their track, course, and drift angles (i.e., their zigzags headed more towards crosswind) with increased dose, but slowed their velocity. No differences in flight track parameters were observed for moths provided with a superimposed pheromone plume, regardless of the background cloud dose. Moreover, moths were able to locate the source of superimposed air plumes in the highest background dose, and of superimposed pheromone plumes in any background dose. The significance of these results is discussed in the context of mating disruption.     

Odor discrimination using insect electroantennogram responses from an insect antennal array

Insects have a highly developed olfactory sensory system, mainly based in their antennae, for the detection and discrimination of volatile compounds in the environment. Electroantennogram (EAG) response profiles of five different insect species, Drosophila melanogaster, Heliothis virescens, Helicoverpa zea, Ostrinia nubilalis and Microplitis croceipes, showed different, species-specific EAG response spectra to 20 volatile compounds tested. The EAG response profiles were then reconstructed for each compound across the five insect species. Most of the compounds could be distinguished by comparing the response spectra. We then used a four-antenna array, called a Quadro-probe EAG, to see if we could discriminate among odorants based on the relative EAG amplitudes evoked when the probe was placed in plumes in a wind tunnel and in a field. Stable EAG responses could be simultaneously and independently recorded with four different insect antennae mounted on the Quadro-probe, and different volatile compounds could be distinguished in real time by comparing relative EAG responses with a combination of differently tuned insect antennae. Regardless of insect species or EAG amplitudes, antennae on the Quadro-probe maintained their responsiveness with higher than 1 peak/s of time resolution.     

Electrophysiological basis for the behavioural response of male and female Trichoplusia ni to synthetic female pheromone

Electroantennogram (EAG) studies demonstrated that antennae of both male and female Trichoplusia ni have: (1) receptor-neurones sensitive to female pheromone, (2) a low response threshold, (3) an identical mean-percentage EAG curve over a broad concentration range of pheromone, and (4) a similar absolute recovery interval from adaptation to pheromonal stimulation. These factors suggest that antennae of male and female T. ni have homologous and homogeneous acceptor sites for the female pheromone. Pheromonal stimulation of female antennae elicited EAGs with only 25% of the amplitude of those elicited in males.