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The recognition that plant volatile signals, long known to be
important in the mediation of plant±parasitoid interactions, also
transmit information to herbivores expands our view of tritrophic
systems and is signi®cant with regard to our understanding of the
selective pressures governing the evolution of such signals. We are
currently exploring whether moths can interpret the herbivorespeci
®c information that these signals convey to parasitoids13 or
rather use these signals only as generalized indicators of insect
feeding. If moths can interpret the higher-order information content
of these signals, then they may be making sophisticated choices
based on the likely presence of particular larval competitors and
perhaps even of particular predators and parasitoids.
Methods
Volatile collection and analysis
All experiments were conducted in an insect-free greenhouse (temperature 2964 8C). Ten
third-instar larvae of either H. virescens, H. zea or M. sexta were allowed to feed
continuously on the leaves of an eight-week-old, greenhouse-grown tobacco plant
(Nicotiana tabacum strain K326) enclosed in a volatile collection chamber24, beginning the
night before collection commenced.
Plant volatiles were collected 24-h a day in 2-h intervals by pulling 1 l of the air passing
over the plant (5 l min-1) through Super Q adsorbent (25 mg) traps at the base of the
volatile-collection chamber; the remainder of the air vented out of the bottom of the
system24. Traps were rinsed with 150ml methylene chloride, 400 ng of n-octane and nonyl
acetate were added as internal standards, and samples were analysed by gas chromatography
and mass spectrometry13 (electron impact and chemical ionization with isobutane
reagent gas). Volatile compounds were identi®ed by comparison of chromatographic
retention times and mass spectra with those of commercially available standards analysed
on the same instruments. Quanti®cation was based on peak area (¯ame ionization
detector) relative to that of internal standards.
Average volatile released by plants from 11:00 to 13:00, 50 h after initial damage:
(ng h-1, s.d.): (E)-2-hexenal (190.93, 23.06); (Z)-3-hexen-1-ol (62.04, 40.08); (E)-bocimene
(5510.46, 789.72); indole (212.67, 45.03); b-caryophyllene (7481.88, 823.40);
a-humulene (221.88, 13.08); (E,E)-a-farnesene (1220.48, 152.81), unidenti®ed sesquiterpene
(1148.25,165.85).
Average volatile released by plants from 19:00 to 21:00, 58 h after initial damage:
(ng h-1, s.d.): (E)-2-hexenal (488.44, 36.78); (Z)-3-hexen-1-ol (792.70, 160.12); (Z)-3-
hexenyl acetate (987.99, 97.12); (E)-b-ocimene (1610.46/349.32); (Z)-3-hexenyl isobutyrate
(123.84, 23.76); (Z)-3-hexenyl butyrate (488.85, 121.67) indole (212.67, 45.03); (Z)-
3-hexenyl tiglate (1328.87, 198.76), b-caryophyllene (2452.88, 223.46); a-humulene
(83.48, 35.17); (E,E)-a-farnesene (888.88, 154.98), unidenti®ed sesquiterpene (960.59,
200.91).
Behavioural assays
Twelve eight-week-old, potted, greenhouse-grown tobacco plants were placed in a
screened cage (4 ´ 2 ´ 3m) with six plants on each side of the cage (80 cm between plants).
On one side only undamaged plants were used, on the other side two of the plants had been
fed on by 10 laboratory-reared third-instar H. virescens (two larvae per leaf) for 48 h.
Larvae were removed from plants before moth release. Three mated H. virescens females
(18 females per treatment) were released in the central sector of the cage at dusk. To
account for a possible directional preference by the moths, two cages were used so in one
the damaged plants were on the north end and in the other they were on the south end of
the cage. The moths' behaviour was visually observed for one hour (from approximately 19:00 to 20:00) and the time (min.) spent by moths on each side of the cage was
recorded. Egg numbers per plant on each side of the cage were counted at 06:00 the next
morning. Similar procedures were used for assays measuring oviposition and behaviour
of female moths in response to plants with or adjacent to synthetic blends, but in this
case plants on both sides of the cages were undamaged. For these treatments, pots of
undamaged plants with volatile-releasing rubber septa on wooden sticks (three per
plant) replaced the damaged plants. We evaluated the response of moths to three
synthetic volatile blends (nocturnal volatiles, exclusively nocturnal volatiles, and diurnal
volatiles). Rubber septa without the synthetic blend were placed on the control side to
neutralize any visual preference. Each bioassay was conducted on three days (two
repetitions each) to account for day-to-day variation. Preference between the
undamaged and damaged plants by the herbivores was subjected to analysis of variance
(an arc-sine square root transformation of the percentage data was used; SAS Institute,
Cary, North Carolina).
Synthetic blends
Synthetic blends were formulated according to a method developed to predict the release
ratio of volatile compounds from rubber septa19,20. Calculations of predicted release ratios
are based on relative vapour pressures19 of the components and the original amounts
released in the natural blend. Blends were dissolved in hexane and 0.3 ml of a blend
solution was pipetted onto a rubber septum. Volatiles released by the blend when
formulated on rubber septa were sampled and analysed. The relative amounts were
adjusted to correct for slight deviations from the predicted amount. Compounds used to
prepare synthetic blends were obtained from Sigma-Aldrich or from Chemical Samples
and analysed by gas chromatography and mass spectrometry to determine purity. All
synthetic compounds were at least 98% pure except for ocimene where both isomers were
present (60% trans and 40% cis).
Release rates of synthetic blends: daytime blend 1, (E)-2-hexenal (200 ng h-1); 2, (E)-bocimene
(5,500 ng h-1); 3, b-caryophyllene (7,300 ng h-1); 4, a-humulene (200 ng h-1); 5,
(E,E)-a-farnesene (1,180 ng h-1). Night-time blend 1, (E)-2-hexenal (500 ng h-1); 2, (Z)-
3-hexen-1-ol (850 ng h-1); 3, (Z)-3-hexenyl acetate (1,000 ng h-1); 4, (E)-b-ocimene
(1,500 ng h-1); 5, (Z)-3-hexenyl isobutyrate (100 ng h-1); 6, (Z)-3-hexenyl butyrate
(500 ng h-1); 7, (Z)-3 hexenyl tiglate (1,200 ng h-1); 8, b-caryophyllene (2,200 ng h-1);
9, (E,E)-a-farnesene (900 ng h-1). Exclusive night-time blend 1, (Z)-3-hexen-1-ol
(850 ng h-1); 2, (Z)-3-hexenyl acetate (1,000 ng h-1); 3, (Z)-3-hexenyl isobutyrate
(100 ng h-1); 4, (Z)-3-hexenyl butyrate (500 ng h-1); 5, (Z)-3-hexenyl tiglate (1,200 ng h-1).