Extreme Sensitivity in an Olfactory System

Anna Maria Angioy 0 2 Alessandro Desogus 0 2 Iole Tomassini Barbarossa 0 2 Peter Anderson 0 1 2 Bill S. Hansson 0 1 2 0 Cagliari , Monserrato-Cagliari , Italy 1 Department of Crop Science, Division of Chemical Ecology, Swedish University of Agricultural Sciences , Alnarp , Sweden 2 Department of Experimental Biology, Section of General Physiology, University of Cagliari , Monserrato-Cagliari , Italy We recorded olfactory-induced cardiac responses to evaluate olfactory response thresholds to behaviourally relevant odours in a moth. Specific antennal receptor neurons enable insects to detect biologically meaningful odours such as sex pheromones and host-plant volatiles. The response threshold values demonstrated here are well below anything earlier reported in any organism. A heart response was triggered by less than six molecules of the most efficient odours hitting the antennae of the insect. The behavioural significance of this extreme sensitivity most likely lies in the creation of awareness and readiness to respond behaviourally at higher concentration levels. - The olfactory system has become an important model system regarding sensory detection and integration. In this context, insects represent an important source of information regarding fundamental principles of olfactory detection (Ziegelberger, 1995; Clyne et al., 1999; de Bruyne et al., 1999, 2001; Vosshall et al., 1999; Strtkuhl and Kettler, 2001) and central nervous information processing (Hansson et al., 1991, 1992; Stopfer et al., 1997; Ito et al., 1998; Christensen et al., 2000; Dubnau et al., 2001; McGuire et al., 2001). Extensive investigations of the anatomy and function of the olfactory system of moths have provided a very useful model for neuroethological studies (Hartlieb and Anderson, 1999). Moth olfactory receptor neurons (ORN) exhibit a high degree of selectivity and sensitivity to both pheromone and non-pheromone volatiles, thus supplying the brain with high-quality information on odour identity, intensity and spatiotemporal distribution (Hansson et al., 1992; Hansson, 1995; Christensen et al., 1996, 2000; Hansson and Christensen, 1999; Vickers et al., 2001). For instance, the high sensitivity to the female-produced sex pheromone exhibited by the silk moth (Bombyx mori) male allows it to respond behaviourally when just 85 ORNs/antenna intercept one molecule each per second (Kaissling and Priesner, 1970; Kaissling, 1971). On the other hand, much higher stimulus intensities are generally reported as minimum values needed for recording significant activity from ORNs (Kaissling, 1987), CNS neurons (Hansson and Christensen, 1999), as well as for driving an appropriate insect behaviour (Todd and Baker, 1999). Odour detection induces cardiac responses that have been described as a sensitive tool for testing insect olfactory reactivity (Queinnec and Campan, 1976). Analogous responses also occur following stimulation of types of sensory receptors, such as visual (Thon, 1982), gustatory (Angioy, 1988) and mechanical (Ai and Kuwasawa, 1995). On the basis of facilitatory influence on motor activity, heart responses to visual stimulation were suggested to play a preparatory role for ensuing behaviour (Thon, 1982). Short cardiac response latencies (<1 s) suggest that sensory input activates a reflex control mechanism (Thon, 1982; Angioy, 1988; Angioy et al., 1987) along cardiac innervation (Davis et al., 2001). In blowflies, an immediate arrest of a fast phase activity and a prompt setting in of a slower one occur after olfactory stimulation with several kinds of volatiles (Angioy et al., 1987). In Heliothis virescens moths, a sudden shift from a low-frequency phase of cardiac activity to a highfrequency one follows stimulation with sex-pheromone or 1-hexanol molecules at concentrations below threshold values eliciting behavioural responses (Angioy et al., 1998). Here we show an extremely high sensitivity of cardiac responsiveness to sex pheromone and plant odour information in both sexes of the cotton leaf worm moth, Spodoptera littoralis, an olfactory sensitivity higher than ever reported before. S. littoralis is a noctuid moth that has been very well investigated concerning olfactory function and olfactory induced behaviour. Specific receptor neurons tuned to the odours used in the present study have been identified on the antenna. Unlike most other moths, the female also possess a well-developed sense for the sex pheromone components produced by herself. The function of this autodetection is so far unknown. Materials and methods Experiments were performed on 25-day-old adults of S. littoralis. Larvae were obtained from the Swedish University of Agricultural Sciences in Alnarp, Sweden and reared on a semi-synthetic diet (Hinks and Byers, 1976) using potatoes instead of beans. Moths were separated by sex at the pupal stage, put into emergence boxes and kept in a cabinet at 24C, 7080% relative humidity, 7/17 night/day cycle. Adults were kept without food, but were provided with water ad libitum during the 24 h prior to experiments. Moths were fixed dorsal side up on a strip of low melting point dental wax; both wings and legs were immobilized. Using soft wet paper, cuticular scales were removed from small areas of the mesothoracic and abdominal dorsal body surfaces to allow positioning of the electrodes. Each moth was placed on a microscope stage in the visual field of a stereomicroscope (Wild M5A; Wild Leitz Ltd, Heerbrugg, Switzerland) within a Faraday shield on an antivibration surface. Cardiac activity recording Monopolar extracellular electrocardiograms (ECGs) were performed on intact specimens using a pair of metal electrodes (AgAgCl wires, 250 m diameter) in contact with the insect cuticle by means of a conductive ECG gel. The active electrode, connected to an amplifier (Altech Electronics, Italy), was positioned on the fourth abdominal segment. The ground electrode was placed on the mesothorax. Signals were displayed on the screen of an oscilloscope (Tektronix 5111A; Tektronix Inc. Beaverton, OR), stored on a modified video recorder (Vetter; A.R. Vetter Co. Inc. Rebesburg, PA) and later analysed with an integrated system of hardware and software (MacLab System; AD Instruments Ltd, Castle Hill, Australia). Olfactory stimulation A main flow of humidified and charcoal-filtered air (1.70 l/min) was continuously delivered through a glass tube (i.d. 8 mm), ending 2 cm in front of the moth antennae. The tip of a Pasteur pipette, containing a 7 15 mm piece of filter paper with (stimulus) or without (control) an olfactory stimulus, was inserted into a small opening in the glass tube, 70 mm from the antennae. By means of a mechanical puffing device (Altech Electronics, Italy), a 1 s air pulse (0.50 l/min) was then sent through the Pasteur pipette. A glass funnel (i.d. 5 cm) connected to an air suction line was positioned close to the preparation to take away the odour-carrying air after stimulati (...truncated)