# Identification of Blackberry (_Rubus fritticosus_) Volatiles as _Drosophila suzukii_ Attractants
## 2 Materials and Methods
### Chemical Analysis of R. fritticosus Volatiles  
The berries of _R. fritticosus_ cv. Lochness originated from a greenhouse at the Research Centre for Fruit Cultivation, pcfruit npo, Sint-Truiden, Belgium (50.462425\({}^{\circ}\) N, 5.93779\({}^{\circ}\) E). The ripe berries were picked when they were completely black. Afterwards, the berries were cut to fit the 20 ml headspace vials and frozen (\(-18\)\({}^{\circ}\)C) until analysis. There were four biological replicates, with each sample containing one berry. To identify and quantify the volatile compounds emitted by _R. fritticosus_, the berry samples were analyzed using MHS-SPME followed by GC-MS (MHS-SPME GC-MS) as in [30; 31; 32]. The sample was kept at 60 \({}^{\circ}\)C under agitation for 5 min using a TriPlus RSH Autosampler (Thermo Fisher Scientific, Waltham, MA, USA). The temperature is increased compared to natural conditions to improve the recovery of the compounds, which is essential for the quantification, while keeping the temperature low enough to avoid artifacts [33]. Following [34; 35], the volatiles were extracted by binding to a 50/30 \(\upmu\)m DVB/CAR/PDMS coating fiber (Supelco, Darmstadt, Germany) and injected using splitless mode with an inlet temperature of 270 \({}^{\circ}\)C, a split flow of 9 mL/min, purge flow of 5 mL/min and a splitless time of 3 min. The flow of helium carrier gas was programmed to start at 2.7 mL/min for 0.1 min after which it slowed down with 20 mL/min\({}^{2}\) until a flow of 0.9 ml/min was reached. The volatiles were separated using a Thermo Trace 1300 GC system (Thermo Fisher Scientific) with an MXT-5 column (30 m length \(\times\) 0.25 mm inner diameter \(\times\) 0.25 \(\upmu\)m film thickness; Restek, Bellefonte, PA, USA) and an ISQ mass spectrometer (Thermo Fisher Scientific). The oven temperature changed at the following rate: it started at 30 \({}^{\circ}\)C for the first 3 min, then it increased with 7 \({}^{\circ}\)C/min until the temperature reached 80 \({}^{\circ}\)C, afterwards, the temperature increased with 2 \({}^{\circ}\)C/min until 125 \({}^{\circ}\)C, after which the temperature increased with a rate of 8 \({}^{\circ}\)C/min till it reached 270 \({}^{\circ}\)C. Mass spectra were recorded in a range of 33-550 amu, using a scan time of 0.2 sec. A mix of linear alkanes (C7-C40 Saturated Alkanes Standard, Supelco, Sigma) were run under the same conditions and served as external calibration to calculate cubic spline interpolated retention indices [36]. From each sample, volatiles were extracted and analyzed over fifteen consecutive runs, by which point most volatiles present in the blackberry sample were exhausted. This MHS-SPME GC-MS method (MHS-SPME GC-MS) allowed us to quantify the total amount of each volatile present in one berry [30; 31; 32]. GC-MS data of the blackberry samples were analyzed as described by [PERSON] et al. [7]. This resulted in 38 unique volatile compounds being identified. In short, CDF files of the chromatograms were analyzed using AMDIS (version 2.71) to deconvolute overlapping peaks. With NIST MS Search (version 2.2), spectra were annotated based on measured retention time, retention index and mass spectral matches to the NIST2011, FFNSC and Adams GC-MS mass spectral libraries. For every individual compound in each sample, the elution profiles were then extracted using weighted non-negative least square analysis [37]. This resulted in calculated peak areas for each compound per sample for each of the fifteen runs. Compounds with peak areas lower than 1000 or peak areas from berry samples that were not significantly higher than in the blank samples were left out. In total, 33 of the 38 identified compounds were commercially available at the time of the experiments. The compounds could be subdivided into five functional groups: thirteen terpenes, eleven alcohols, five aldehydes, two esters and two ketones. To estimate the quantity of each compound in one _R.