Chem. Senses 24: 423-428,
1999
© Oxford University Press 1999
Actographic Analysis of the Effects of an Esterase Inhibitor on Male Moth Responses to Sex Pheromone
INRA Unité de Phytopharmacie et Médiateurs Chimiques, Route de Saint Cyr, F-78026 Versailles, France 1 SYNTECH VDP Laboratories, PO Box 1547, 1200 BM Hilversum, The Netherlands and 2 Department of Biological Organic Chemistry, CID (CSIC), Jordi Girona 18–26, E-08034 Barcelona, Spain
Correspondence to be sent to: Dr Michel Renou, INRA Unité de Phytopharmacie et Médiateurs Chimiques, Route de Saint Cyr, F-78026 Versailles, France. e-mail:renou{at}versailles.inra.fr
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Abstract |
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The effects of 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), a trifluoromethyl ketone that inhibits antennal esterases, on male Mamestra brassicae responses to the main pheromone component have been investigated using an actograph. This actograph used a movement detector based on the Doppler effect. The signal from the detector was digitalized and analysed on a PC microcomputer to quantify male activity. When added to the air flowing through the observation chamber, OTFP inhibited the responses of male moths to the pheromone. The number of males responding to the pheromone and the intensity of the response were decreased by OTFP. The latency of the response was increased and its duration decreased. These effects on the kinetics of the behavioural response cannot be directly correlated to the inhibition of pheromone catabolism by OTFP and other targets must be involved. The high level of inhibition of behaviour observed in presence of OTFP demonstrates the interest of trifluoromethyl ketones as mating disruption agents for pest control.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussion and conclusionReferences |
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Male moth behaviour in response to their specific sex pheromone has been intensively investigated using a variety of experimental set-ups (Baker and Cardé, 1984
; Wyatt, 1997). Specifically designed olfactometers and wind
tunnels have been widely used to measure orientation in a pheromone plume (Baker
and Linn, 1984). Qualitative observations are essential to divide the behaviour into
units to records, such as take off, wing fanning, landing at the source, copulation attempts.
Scoring of these elementary responses are used to quantify the level of activity triggered by a
chemical stimulus. Further refinement of the method resulted from tracking walking or flying
insects with a video camera, or recording locomotory paths with a servosphere (Kramer, 1975) or other locomotion compensators, combined with the automatic
analysis of tracks by computer-assisted trajectometry. All these methods require a sophisticated
and expensive equipment and/or demand a large number of insects to achieve quantification of
behaviour.
In the course of our studies on the effects of pheromone analogues on olfaction (Parrilla and Guerrero, 1994; Renou et al., 1997) we
needed a simpler device to screen candidates for inhibition of pheromone communication in
insects. The Doppler radar has already been successfully employed in the monitoring of
spontaneous activity rhythms of flies and cockroaches (Buchan and Satelle, 1979; Buchan and Moreton, 1981). Thus, we designed a simple
recorder using a cheap movement detector, also based on Doppler radar. In this paper we
describe the system and its use to record moth responses to a chemical stimulus. The movements
of the insect are converted in an analogue electrical signal by the radar detector with a high signal
to noise ratio. This signal is connected to a computer via an analogue/ digital card and analysed
with dedicated software. The radar detector has been used to measure the responses of male Mamestra brassicae to the main component of the sex pheromone, (Z)-11-hexadecenyl acetate (Z11-16:Ac) (Descoins et al.,
1978)
either in pure air or in air containing the esterase inhibitor 3-octylthio-1,1,1-trifluoropropan-2one
(OTFP). In vitro, OTFP is a very potent inhibitor of the antennal esterases of the
Egyptian armyworm, Spodoptera littoralis (Duran et al., 1993), enzymes responsible for the catabolism of the pheromone (Quero, 1996). In vivo, OTFP has been
shown to alter pheromone detection (Renou et al., 1997; Pophof, 1998) and to inhibit
behaviour in the wind tunnel after conditioning males to a OTFP-saturated atmosphere. In the
present study we show that an aerial background of OTFP during the test also affects the
response to the pheromone. The effects of OTFP on the response kinetics are commented.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsDiscussion and conclusionReferences |
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Insects
Mamestra brassicae were reared in the laboratory on an artificial medium. Male and female pupae were sorted and kept separately. Adult male moths were provided with a 10% sucrose solution and conditioned to a 16:8 photoperiod. Behavioural tests were performed on 3–5 day old males, during the last 3 h of the scotophase.
Compounds
OTFP was obtained by alkylation of the corresponding thiol with
3-bromo-1,1,1-trifluoropropan-2-one (Parrilla et al., 1994).
HPLC-purified Z11-16:Ac, free of alcohol, was prepared in the laboratory. Dilutions in hexane of
both compounds were obtained at the appropriate concentrations.
Experimental set-up
To establish a homogeneous field of odourized air and to prevent the insect from moving out of the radar beam an observation chamber was made of a glass tube, 120 mm in length x 30 mm i.d., with an upwind end of smaller diameter (6 mm i.d.) and connected to a permanent source of charcoal-filtered air (85 l/h). A lateral branch, perpendicular to the main branch, was used to introduce a Pasteur pipette containing a piece of filter paper loaded with the pheromone. Air from the observation chamber was evacuated out of the room by an exhaust fan. The set-up (Figure 1) was enclosed in a cabinet installed in a climate room at 25°C. Red light provided by a 60 W incandescent lamp positioned above the observation chamber was adjusted at 0.3 lux. Stimulation was achieved by applying an air puff (3 s, 21 ml) through a Pasteur pipette containing a filter paper loaded with 0.1 µg of Z11-16:Ac. After each test, the observation chambers were washed overnight with a 5% solution of Decon (Prolabo, Paris), rinsed in distilled water and dried at 110°C, before being used again.
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The Doppler radar sensor (Alpha Industries, Type GOS2780) had a working frequency of 24 GHz and an output power of 3 µW. The output signal was amplified x10 by a specially designed AC amplifier. The raw analogue signal was high-pass filtered, amplified x10 and fed into an acquisition board IDAC-02 (Syntech, Hilversum) in a PC-based microcomputer. The board had a 16 bit A/D converter, a software controlled amplifier, programmable DSP and input–output FIFO buffer memory. Data acquisition was performed at 500 samples/s during 15 min. To save memory space, software integration enabled us to save only 10 samples/s into a file on the hard disk. The software EAG for Windows (Syntech, Hilversum) was used to edit the recordings and to make direct measurements of the response parameters. Preliminary calibration experiments showed that maximum sensitivity was reached when the radar beam was focused perpendicularly to the chamber, the detector being placed in front of the middle of the observation chamber, 25 mm from the wall. These experiments showed that the amplitude of the signal from the radar depended on the intensity of the movement, its direction and the position of the insect relative to the radar beam. Positive peaks corresponded to movements of the insect toward and negative peaks away from the radar probe. Thus, the male locomotory activity could be easily quantified by summating the absolute values of the signal amplitude over the time.
Bioassays
A single male was introduced into a clean observation chamber and left to acclimate for 1 h
in
the experimental room. Then, OTFP-loaded air was applied into the chamber by introducing a
filter paper loaded with the appropriate amount of OTFP, diluted in hexane, into the air inlet
tube.
After a minimum time of exposure to OTFP of 180 s, a 3 s puff of pheromone was applied. The
insect movements were continuously recorded during the pre-stimulation period and 600 s after
stimulation with Z11-16:Ac. Control experiments were performed using a filter paper loaded
with
pure hexane as a treatment and a puff of Z11-16:Ac as a stimulus. The following parameters
were
measured (Table 1): percentage of males activating at OTFP onset, mean
latency of male activation after Z11-16:Ac stimulus, and mean amplitude of activity over 60 s
and
300 s following the puff of Z11-16:Ac. The behaviours of male moths that initiated their activity
later than 60 s after the puff of Z11-16:Ac were not considered as positive responses. Thus, the
percentage of positive responses to Z11-16:Ac is given inTable 1 and the
behaviour of male moths that met this criterion is analysed separately inTable 2. Data were submitted either to
2 tests for number of responses or
to Kruskal–Wallis analyses for duration and amplitude of responses.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsDiscussion and conclusionReferences |
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Responses to Z11-16:Ac in pure air
When stimulated with a puff of air from a source loaded with 0.1 µg of Z11-16:Ac, male M. brassicae became very active, performing wing fanning and intense locomotion in the observation chamber. Both activities are typical of male moth precopulatory behaviour and they were easily detected by the radar sensor, producing bursts of high-amplitude spikes (Figure 2). The male responses were recorded simultaneously with video and the radar actograph to determine if different behaviours produced different signals. When the insect was immobile the background signal amplitude was <0.5 mV. Movements of antennae, wings or legs produced signals of <1 mV. Displacements in the observation chamber produced signals comprised between 2 and 5 mV whereas the signal could reach 12 mV when locomotion was associated to wing fanning.
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Most of the 58 male moths became active within a few s after the puff of Z11-16:Ac (n = 45, 77.6%). Nine males (15.5%) exhibited an intense activity after 60 s and four insects (7.0%) did not respond at all to the pheromone puff. The average amplitude of activity during the 60 s following the puff of Z11-16:Ac was 73.7 (Table 1, control). Males maintained a high level of activity for several minutes after the stimulus, as shown by the mean amplitude calculated over 300 s (60.4).
Behaviours of male moths that initiated their activity after 60 s were not considered as positive responses to the pheromone and temporal response patterns were analysed separately (Table 2) in the 45 male moths that were activated within 60 s after the puff of Z11-16:Ac. The mean latency of response in these males was of 5.7 s (Table 2). Furthermore, a male moth was considered to have completed its response when it was inactive for >60 s. According to these two criteria, the mean duration of the response to Z11-16:Ac in air was 317.5 s (Table 2). The mean activity amplitude during the whole response was 75.1. Short pauses within a response enabled the discrimination of individual bursts of activity (mean number: 4.3). The first burst of activity was generally longer than the subsequent one (mean duration: 158.8 s). The mean activity amplitude during this first burst reached 109.1.
Responses to OTFP
The introduction of variable concentrations of OTFP into the airflow elicited locomotory
behaviour on males. At doses of OTFP between 1 and 100 µg, 67.9 to 96% of males were
active during the pre-test period (Table 1), versus 18.4% in control
insects exposed to pure air. At 0.1 µg, the percentage of males exhibiting pre-test activity
was 36.7%, a value still significantly different from control (2 = 24.36, P < 0.01).
Responses to Z11-16:Ac in the presence of OTFP
Male responses to a puff of Z11-16:Ac were measured in OTFP-loaded air. Pheromone triggered activity was strongly reduced in the presence of OTFP, as shown by significantly decreased amplitude of activity within 60 and 300 s after the stimulus (Figure 2 andTable 1). The number of male M. brassicae becoming active within 60 s after the pheromone puff (positive responses to Z11-16:Ac) was significantly reduced in the presence of OTFP. With 0.1 µg of OTFP most of the males still responded to the pheromone (80.0%); but with 1 and 100 µg of OTFP the numbers of positive responses were significantly smaller than the control, <30% of males showing post-stimulus activity (Table 1). With 10 µg of OTFP, 56% of males showed post-stimulus activity. How ever, the activity was low (19.5) and brief (74.7 s). Thus, this high score of males showing activity is probably due to the difficulty in discriminating between OTFP- and pheromone-triggered activities, OTFP itself triggering intense locomotory activity in the males.
The temporal characteristics of the response to Z11-16:Ac were also modified in the presence of OTFP. First, the latency of the response was significantly longer with 0.1, 10 and 100 µg (Table 2). Secondly, the duration of the first burst and the overall duration of the response were shorter than in control insects. Thirdly, the mean amplitude during the first burst and the overall mean amplitude were lower.
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Discussion and conclusion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussion and conclusionReferences |
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The radar sensor presents a number of advantages that make it an appropriate and interesting tool not only to monitor sustained spontaneous activity (Buchan and Satelle, 1979
; Buchan and Moreton, 1981) but also to record transient
responses to stimulus. This is particularly true when the temporal patterns of the response are
features of interest. First, it can work at low light intensity or in complete darkness, and the radar
beam can pass through glass and plastic walls of insect containers with no interference regardless
of the orientation of the beam with respect to the wall. Secondly, it can be focused on the target
insect and has a low sensitivity to noise. Thirdly, it is cheap, simple and easy to handle.
Moreover,
it can provide very reliable recordings of the timing and sequence of the response, and can be
used
either with single individuals or with several insects. Further development aiming to improve
quantification of the male responses is in progress.
The behavioural responses of male M. brassicae to the pheromone main component
were reduced or even suppressed in the presence of the trifluoromethyl ketone OTFP in the air.
These effects were dose-dependent. These results are consistent with former experiments in a
wind tunnel that showed inhibition of the flight of male Spodoptera littoralis to a
pheromone source after a 4 h exposure to air with a high concentration of OTFP (Quero, 1996). In the experiments presented here the inhibitory effects arose after a
brief exposure and at lower concentrations of OTFP. Electrophysiological investigations (Renou et al., 1997; Pophof, 1998) have
shown that OTFP, like other trifluoromethyl ketones analogues of pheromone, inhibits the firing
responses of the olfactory receptor neurones to pheromone components in different moth species,
including M. brassicae. Thus, the effects of OTFP on behaviour are most probably due
to
its inhibitory activity on the peripheral sensory system. The high level of inhibition of behaviour
observed in the presence of OTFP demonstrates the interest of trifluoromethyl ketones as mating
disruption agents for pest control.
Flying male moths following an aerial pheromone trail exhibit very fast reaction times to
fluctuations in pheromone concentration (Baker and Vickers, 1995). This
outstanding capacity relies both on the functional ability of the olfactory neurones to resolve
repetitive stimulus rates (Rumbo and Kaissling, 1989) and on efficient
mechanisms of pheromone deactivation (Vogt et al., 1985). Any
factor altering one of these two mechanisms should also strongly affect moth behaviour. Thus,
we
expected that OTFP, which inhibits in vitro the degradation of pheromone components
with an acetate function by the esterases contained in antennal extracts (Parilla and
Guerrero, 1994; Parilla et al., 1994), would increase
in vivo the duration of the behavioural response. However, besides its effects on
response amplitude, OTFP increased the latency of the response to Z11-16:Ac and strongly
reduced the duration of the first burst and of the response. These two effects cannot be directly
correlated to the inhibition of pheromone catabolism by OTFP, so other mechanisms must be
involved. Potential molecular targets for OTFP are the receptors and pheromone binding proteins
(PBP). Evidence for a reaction of OTFP with PBPs comes from in vitro binding
experiments that show competition between tritiated Z11-16:Ac and its TFMK analogue for the
binding sites of the PBPs (P. Nagnan, unpublished data). In vivo, due the role of PBPs
as
carriers for pheromone molecules, such a competition would reduce the availability of the
stimulus for the receptor sites, resulting in a decrease of the response.
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Acknowledgments |
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We thank Dr Jean-Pierre Rospars, Dr Arthur Vermeulen and Dr Pascal Chalande for helpful discussions, Dr Philippe Lucas for a critical reading of the manuscript, Mrs Martine Lettere for preparing Z11-16:Ac and Mr. Taylor Quadjovie for rearing the insects.
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References |
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TopAbstractIntroductionMaterial and methodsResultsDiscussion and conclusionReferences |
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Baker, T.C. and Cardé, R.T. (1984) Techniques for behavioral bioassays. In Hummel, H.E. and Miller, T.A. (eds), Techniques in Pheromone Research. Springer Verlag, New York, pp. 4573.
Baker T.C. and Linn, C.E., Jr (1984) Wind tunnels in pheromone research. In Hummel, H. E. and Miller, T.A. (eds), Techniques in Pheromone Research. Springer Verlag, New-York Berlin Heidelberg Tokyo, pp. 75–110.
Baker, T.C. and Vickers, N.J. (1995) Pheromone-mediated flight in moths. In Cardé, R.T. and Minks, A.K. (eds), Insect Pheromone Research: New Directions. Chapman & Hall, New York, pp. 248–264.
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Accepted April 16, 1999
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