**4. Gustatory extracellular recording methods**

Randomly selected, fifth instar, 12-24 hours post-molt, *Lymantria dispar* larvae, reared on artificial diet (12h light: 12h dark; ca., 60% relative humidity) (Shields et al., 2006) were used for all experiments. The larvae were food deprived at least 12 hours prior to the experiments and were naive to the test compounds. Recordings were carried out at ambient temperatures (24-26ºC). Each larva was transected just behind the head and a blunt-tipped saline-filled glass electrode was inserted into the head with sufficient force to cause the eversion of the lateral and medial styloconic sensilla. This pipette was filled with an electrically conductive solution (typically KCl or NaCl) and served as the ground or indifferent electrode (Figs. 5, 6). The cut end of the head was sealed with a minimal amount of melted bee's wax (Fig. 5A). The stimulating or recording electrode was filled with a test solution and was positioned over the tip of a single styloconic sensillum (Figs. 5A, 6) with the aid of a micromanipulator (Leitz) under visual control on a vibration-free air table (Fig. 5B). The preparation lasted, on average, one to two hours.

Neurophysiological Recording Techniques Applied to Insect Chemosensory Systems 133

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

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),

of the sensillum. This figure was taken from Shields and Martin, 2010.

while jimsonweed provides a nectar feeding source.

Fig. 5. (A) Higher magnification view of the insect preparation. (B) Gustatory extracellular recording set-up. Fig. (A) was modified from Shields and Martin, 2012.
