Signal grass, Brachiaria brizantha
(A.Rich.) Stapf (Poaceae), an important forage crop in Africa, was grown from root splits in pots in a screen house (natural light conditions, approx. L 12 : D 12 h) at ICIPE in Western Kenya. Spotted stemborer, Chilo partellus
Swinhoe (Lepidoptera: Crambidae), moths were reared from larvae collected from the wild, principally sorghum fields, and fed on a natural diet of sorghum cv. Serena. Cotesia sesamiae
Cameron (Hymenoptera: Braconidae wasps, larval parasitoids of C. partellus
) were reared as described by Overholt et al. (1994)
Treated plants were caged with five gravid C. partellus
overnight the night before headspace sampling while control plants were kept in identical conditions in empty cages. Collections began in the last 2 h of photophase the following day. Volatile compounds were collected for 48 h periods from intact plants using standard methods (Agelopoulos et al. 1999
). Foliage (one plant per collection; mean weight = 20.6 g) was enclosed in polyethyleneterephthalate bags (volume, 3.2 l). Charcoal filtered air was pumped in and air was drawn out at 400 ml min−1
through Porapak Q (0.05 g) filters.
Collected volatiles were eluted from Porapak Q with diethyl ether and analysed on a capillary gas chromatography (GC) column (50 m × 0.32 mm ID, HP-1) directly coupled to a mass spectrometer (Autospec Ultima, Micromass, UK). Ionization was by electron impact at 70 eV, 250°C. Oven temperature was maintained at 30°C for 5 min, then programmed at 5°C min−1 to 250°C. Authentic (Z)-3-hexenyl acetate (Z3HA) (99% purity, Avocado Research Chemicals Ltd., Lancs, UK) was used as an external standard to calibrate GC peak area and quantify the amount present in each headspace sample.
Electroantennogram (EAG) recordings were made from female C. sesamiae
and replicated five times. An antenna was excised and suspended between two Ag–AgCl glass electrodes filled with Ringer saline solution. Responses were measured in millivolt deflections and signals passed through a high impedance amplifier (UN-06, Syntech, The Netherlands). The coupled GC-electrophysiology system, in which effluent from a GC column is simultaneously delivered to the antennal preparation and a GC detector, has been described (Wadhams 1990
). Separation of volatiles was achieved using an Agilent 6890 GC fitted with a non-polar column (HP-1, 50 m × 0.32 mm ID × 0.52 µm film thickness). Oven temperature was maintained at 40°C for 2 min, then programmed at 5° min−1
to 100°C and then at 10°C min−1
to 250°C. Simultaneous records of the EAG and flame ionisation detector responses were obtained with commercial software (Syntech, The Netherlands).
A Perspex four-arm olfactometer (Pettersson 1970
) was used with air drawn through the four arms towards the centre at 350 ml min−1
. Mated female C. sesamiae
wasps, without previous exposure to plants or prey, were transferred individually into the central chamber. Time spent in each arm was recorded using OLFA (Udine, Italy) software over a 10 min period. Aliquots of 10 µl of headspace samples (dose similar to that emitted by 12 plants over 10 min) were applied to a filter paper strip inserted into an inlet port at the entrance of an arm. A choice test was carried out: one arm had a headspace sample from plants with oviposition while the opposite arm had a sample from clean plants. The remaining two arms were blank controls (solvent only).
In an oviposition bioassay, two four-week-old potted plants, one clean (no prior oviposition) and one with eggs (caged with five gravid female C. partellus moths the night before), were placed in a cage (80 × 40 × 40 cm) covered by a fine wire mesh netting. The following night, another five gravid female C. partellus moths were introduced into each cage and allowed to oviposit overnight. Plants were removed and the number of fresh eggs on each plant counted under a light microscope at ×6.5 magnification. Fresh eggs were distinguished by a difference in colour.
Data on oviposition preference were analysed with a two-sample t
-test and mean times spent in olfactometer arms were compared by one-way analysis of variance (ANOVA) after confirming that data were normally distributed. Means were separated using Tukey's studentized range test. All analyses were performed using SAS (SAS Institute 2001