Before the collection of volatiles, the fiber was preconditioned in the GC injection port at 250 °C for 20 min and then inserted into conical flask through the septum, afterwards exposed to the headspace for 40 min at 60 °C in a water bath.
The synthetic blends were used here, rather than a collection of volatiles from nature, as the full range of inhibitory candidates and attractants of interest for the responses of the predator are not available from any single biological source.
Steel sorbent cartridges (89 mm ×6.4 mm O.D.; Markes) containing 200 mg Tenax TA 20/35 for collection of volatiles were conditioned for 40 min at 280°C under a nitrogen flow of 20 psi using a TC-20 multi-tube conditioner and were kept airtight with brass caps until use.
There is no agreed standard for the collection of breath for volatile analysis and uncontrolled breath sampling has been shown to be unreliable (Schubert et al., 2001; O'Hara et al., 2008).
Here, we show that our method of volatile collection does not alter tissue morphology.
The parameters such fiber coating (75 µm PDMS/Carboxen Cat No. 57284-U and 65 µm PDMS/DVB), sample size (2- and 3-mm) as well as post biopsy time of volatile collection were investigated (Figure S1).
Regarding floral volatiles, their emission was increased after Pieris infestation but, because the number of open flowers at the time of volatile collections was not counted, it is possible that the relative amount of floral volatiles released per flower remained stable or even decreased.
Volatile samples were analyzed within 4 days of volatile collections by a gas chromatograph (GC, Agilent 6890 N) connected to an HP5 column (30 m × 0.32 mm internal diameter × 0.25 μm film thickness) and an Agilent 5975 mass selective detector associated to a thermal desorption system (TDS2, Gerstel, Mühlheim Germany).
All wood samples were placed in a freezer within 4 h of collection to avoid the loss of volatile organic compounds.
This allows collection of all steam and volatile components (furfural and acetic acid) in the slurry for more accurate component mass balance measurements.
