**5. Olfactory stimuli and odor delivery**

Olfaction is the principal sensory modality through which insects locate their food sources, mates, and oviposition sites. Over the past three decades, or so, moth olfaction has focused primarily on mechanisms through which male moths detect, process sensory information about, and respond behaviorally to the sex pheromones emitted by conspecific females. In contrast, relatively little is known about similar mechanisms with respect to nonpheromonal odors, such as plant volatiles and the mechanisms by which female moths detect and discriminate plant-associated volatiles (odorants) for foraging and oviposition purposes and how this information is processed by the olfactory system.

Previous studies have indicated that scent emitted by sphinx moth-pollinated flowers (Knudsen and Tollsten, 1993; Raguso and Willis, 1997; Raguso and Light, 1998), as well as floral and vegetative volatiles of tobacco, tomato, and other hostplants (Andersen et al., 1988; Buttery et al., 1987a; 1987b) attract adult *M. sexta* for feeding (Morgan and Lyon, 1928; Yamamoto et al., 1969; Raguso et al., 1996; Raguso and Light, 1998). Flowers having a "white floral" scent, such as that associated with many night-blooming moth-pollinated flowers, possess acyclic terpene alcohols (e.g. linalool, nerolidol, and farnesol), as well as the corresponding hydrocarbons, aromatic alcohols, and esters derived from them, in addition to

Fig. 5. (A) Higher magnification view of the insect preparation. (B) Gustatory extracellular

Olfaction is the principal sensory modality through which insects locate their food sources, mates, and oviposition sites. Over the past three decades, or so, moth olfaction has focused primarily on mechanisms through which male moths detect, process sensory information about, and respond behaviorally to the sex pheromones emitted by conspecific females. In contrast, relatively little is known about similar mechanisms with respect to nonpheromonal odors, such as plant volatiles and the mechanisms by which female moths detect and discriminate plant-associated volatiles (odorants) for foraging and oviposition

Previous studies have indicated that scent emitted by sphinx moth-pollinated flowers (Knudsen and Tollsten, 1993; Raguso and Willis, 1997; Raguso and Light, 1998), as well as floral and vegetative volatiles of tobacco, tomato, and other hostplants (Andersen et al., 1988; Buttery et al., 1987a; 1987b) attract adult *M. sexta* for feeding (Morgan and Lyon, 1928; Yamamoto et al., 1969; Raguso et al., 1996; Raguso and Light, 1998). Flowers having a "white floral" scent, such as that associated with many night-blooming moth-pollinated flowers, possess acyclic terpene alcohols (e.g. linalool, nerolidol, and farnesol), as well as the corresponding hydrocarbons, aromatic alcohols, and esters derived from them, in addition to

recording set-up. Fig. (A) was modified from Shields and Martin, 2012.

purposes and how this information is processed by the olfactory system.

**5. Olfactory stimuli and odor delivery** 

esters of salicylic acid (Knudsen and Tollsten, 1993). Also thought to play a significant role in the attraction of insects to, and their recognition of, their hostplants are various plantassociated odorants, such as green-leaf volatiles (mainly saturated and unsaturated C6 alcohols and aldehydes), terpenoids, and benzenoid compounds (e.g. Boeckh, 1974; Visser and Avé, 1978; Renwick, 1989; Heath et al., 1992; Knudsen and Tollsten, 1993; Raguso and Willis, 1997).

Fig. 6. Schematic diagram showing extracellular recording from a single gustatory styloconic sensillum showing the stimulating electrode containing the stimulus positioned over the tip of the sensillum. This figure was taken from Shields and Martin, 2010.

The majority of the selected, purified, and synthetic odorants tested (chemical purity of 95- 99.9%) (Shields and Hildebrand, 2001a) represented floral headspace volatiles of native, night-blooming flowers, such as *Datura wrightii* (jimson weed), *Hymenocallis sonorensis* (spider lily), and *Oenothera caespitosa* (evening primrose), to which *M. sexta* and other sphinx moths are attracted for nectar-feeding and were available from Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI). Complex blends of volatiles emitted by the headspace inflorescence from *D. wrightii* (i.e., jimsonweed) and the foliage of two solanaceous plants (i.e., *Lycopersicon* (tomato) and *Nicotiana* (tobacco) species were also tested. This was carried out by placing the entire inflorescence or two to three undamaged leaves in a 20 ml capped syringe, which was allowed to equilibrate at room temperature for 24 h prior to use. Tomato and tobacco foliage are preferred by female *M. sexta* for oviposition (Yamamoto et al., 1969), while jimsonweed provides a nectar feeding source.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 135

Fig. 7. Representative extracellular olfactory electrophysiological recordings of from three different olfactory receptor cells (ORCs) from female *Manduca sexta* trichoid type-A sensilla showing excitatory (A-F, H-M) and inhibitory (G) responses. The responses in (F) and (G) are from a different ORC than those in (A-E) and (H-M). The stimulus bar is represented by a filled rectangle and represents 200 ms and represents the onset of odor delivery. This

figure from modified from Shields and Hildebrand, 2001b.

For testing individual compounds, each odorant was dissolved in odorless mineral oil (light white oil, Sigma, St. Louis, MO) at a dilution of 1:10 (v:v). A 30-µl aliquot was pipetted onto a 1.5-cm2 piece of grade-1 filter paper (Whatman, Kent, ME) and inserted into a disposable 20 ml syringe. Control syringes, loaded with 30 µl of mineral oil, were also prepared. All syringes were capped and allowed to equilibrate, similar to that described above. After each experimental session, the capped odorant syringes were stored at 4°C. Some odorants eliciting the strongest responses were tested in dose-response experiments. For these experiments, dilutions were prepared in decadic steps (v:v) over at least four log units from stock solutions. Stimulus loads ranged from 3 x 10-4 to 3 μl of the test compound (approximately 0.3 μg to 3 mg). Odorants were tested in order of increasing concentration to prevent adaptation of the ORCs. Also included among the odorants, were two key components of the female moth's sex pheromone, E10,E12-hexadecadienal (bombykal) and E11,Z13-pentadecadienal ("C15"), a relatively stable mimic of E10,E12,Z14-hexadecatrienal (see below) (Kaissling et al., 1989). Each compound was dissolved in *n*-hexane and applied directly (without mineral oil) at 1 ngμl-1 and 10 ngμl-1 to the filter paper.

The odor was delivered to the female antenna using a stimulating device (type CS-01, Syntech) (Shields and Hildebrand, 2001a). The tip of the syringe containing the odorant was introduced into a 20 cm long, L-shaped glass tube (1 cm i.d.) whose open end was positioned 5-7 mm from the test antenna (Fig. 3). The end of the tube was flattened to 0.5 cm to correspond with the length of the antenna. The antenna was continuously flushed with a 5 cms-1 stream of charcoal-filtered, humidified air. To deliver a stimulus, 2 ml of odor-laden air was injected at a constant rate over 200 ms from the syringe into the airflow using a motor-driven syringe olfactometer, resulting in increased airflow of 20 cm s-1 (measured by a thermo-anemometer) (Fig. 3). A glass funnel (3.5 cm i.d.) attached to an air evacuation line was positioned near the preparation to draw away any odor-bearing air following stimulus delivery. Odor stimuli were presented randomly to the sensilla to be tested. Blank controls were tested repeatedly, but never elicited a response from the ORCs. An interstimulus interval of at least 60 s, or longer, if necessary for spontaneous activity of the ORC to return to their initial value, was allowed between stimulations.
