# Identification of Blackberry (_Rubus fritticosus_) Volatiles as _Drosophila suzukii_ Attractants
## 2 Materials and Methods
### Experimental Set-Up  
To measure the attractiveness of each of the blackberry volatiles, a two-choice trap-based bioassay was used. Gravid female flies (4-10 days old) were used in the experiment. Males and female flies were anesthetised using CO\({}_{2}\) and separated based on the presence or absence of the black wing spot, characteristic for _D. suzukii_ males. The set-up consisted of three cylindrical polystyrene containers (Greiner Bio-one, container for plant tissue culture). The top of the middle container was closed off with a mesh, which allowed for sufficient air ventilation and prevented saturation inside the set-up. The outer containers were closed with a coenzyme stopper (Greiner Bio-one, Kremsmunster, Austria). The containers were connected with a 6.5 cm PTFE tube (6.35 mm ID \(\times\) 7.94 mm OD, Cole-Parmer, Vernon Hills, IL, USA) (Figure 1). The tube connecting the containers was made to stick out 2 cm in the outer containers in order to create a trapping effect once the fruit fly made a choice [15]. A moist cotton pad was placed at the bottom of the three containers to prevent the flies from dehydrating. At the start of each experiment, around 30 flies were placed in the middle container. This container included a 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany) with water and cotton wool to ensure a constant supply of water. The middle container also had a strip of red tape, as odors and color are known to be able to synergize [38] and preliminary experiments showed that this increased the response of the fruit flies. Subsequently, the flies were allowed to make a choice between the treatment and control arms in the bioassay over a total period of 24 h, while being placed under a 16:8 light:dark regime at 25 \(\pm\) 1 \({}^{\circ}\)C. All containers were put in randomized orientation in open boxes with the sides covered to prevent any systematic biases in preference.  
When the experiment was finished, the containers were placed in the freezer and all flies were manually counted. From these counts, we then calculated attractivity, which was defined as the proportion of flies that chose the treatment vs. the control side, as well as a choice factor, which was defined as the proportion of flies that made a choice (i.e.,  
Figure 1: Experimental set-up of the two-choice trap-based bioassays that was used to test the attractiveness of identified _R. fruitcossus volatiles_. The set-up consisted of three containers with a moist cotton pad placed on the bottom to provide sufficient humidity. At the start of each experiment, around 30 flies were released in the middle container, which contained ad libitum water and which was connected to the outer containers via tubes with a length of 6.5 cm. The outer container either contained an Eppendorf tube with a volatile compound identified from _R. fruitcossus_ (in an amount corresponding to that estimated to be present in 10 berries and dissolved in 100 uL of mineral oil) (treatment arm) or mineral oil solvent only (control arm). After 24 h, the overall attractiveness was assessed based on the number of flies present in each container.  
choosing either the treatment or control container vs. those that made no choice, staying in the middle container). In between subsequent experiments, all containers and tubes were thoroughly cleaned with 70% ethanol and placed in an oven at 60 \({}^{\circ}\)C overnight to ensure that any remaining volatiles had evaporated. In principle, a volatile compound that showed a high and statistically significant attractive effect and also displayed a high choice factor was expected to potentially serve as a good lure.