Gc grade

Headspace volatiles targeting the vegetative parts were collected from four of the identified host plants fed on by sand flies (~70% of samples analyzed) based on plant DNA detection namely, V. tortilis, S. laeta, V. nilotica and P. juliflora (see results section). This was carried out in situ in their natural habitats at the study site, using portable field pumps (Analytical Research System, Gainesville, Florida, USA). Odors from the four plants were collected by enclosing a vegetative part in an airtight oven bag (Reynolds, Richmond, VA, USA) (S1 Fig) by passing charcoal-filtered air at a flow rate of 350 ml/min on to two Super-Q adsorbents (30 mg, Analytical Research System, Gainesville, Florida, USA) for each replicate substrate. For all the plant species, volatiles were collected for 12 hr (06:00–18:00) during the day and 12 hr (18:00–06:00) at night and replicated three to four times for each plant species using a different plant. The same procedure was applied for the control, comprising volatiles from blank oven bags in the same habitat of the target plant. The Super-Q traps were each eluted with dichloromethane (DCM) (200 μl) (GC-grade, Sigma Aldrich, Gilling-ham, UK) and analyzed using coupled gas chromatography/mass spectrometry (GC-MS).

Then samples were lysed using a Precellys bead-mill with 3 cycles at − 10 °C, 6500 rpm for 15 s and 10 s breaks in between runs. The mix was centrifuged (20,000×g, 4 °C, 15 min) and its supernatant subsequently dried in a vacuum centrifuge (Eppendorf, Hamburg, Germany). Dried samples were derivatised with 20 µL methoxyamine (20 mg/mL in Pyridine) at 28 °C for 90 min under constant shaking. Afterwards, samples were derivatized with MSTFA (N-Methyl-N-(trimethylsilyl)trifluoroacetamide; 37 °C, 30 min) and directly analyzed afterwards. QC samples were prepared by collecting and pooling 5 µL from each sample. The mixture was vortexed and aliquoted. A mixture of n-alkane mix (C₁₀–C₄₀, even; Neochema, Bodenheim, Germany) in hexane (GC-grade, Sigma-Aldrich, Taufkirchen, Germany) was used for retention index calculation.

For transesterification, the dried isolates were incubated for 16 h in 1 N methanolic boron trifluoride (MeOH/BF₃; Fluka) at 70°C. Saturated NaCl was added to stop the transesterification reaction and to advance the following phase separation. Dotriacontan (C₃₂ alkane, 10.025 mg / 50 ml) was added as internal standard to each sample. The soluble hydrophobic components were extracted by adding chloroform. The chloroform phase was transferred to a new vial and extraction was repeated three times. The extract was dried with water free Na₂SO₄ and the volume was reduced to 50 μl by evaporation under N₂ flow.
Samples were derivatized in 20 μl BSTFA (N,N-bis(trimethylsilyl)-trifluoracetamide; Machery-Nagel, Düren, Germany) and 20 μl dry pyridine (GC-grade, Merck, Darmstadt, Germany) for 40 min at 70°C. Pyridine catalyzed the derivatization reaction and BSTFA masked free hydroxyl- and carboxylgroups forming the corresponding trimethylsilyl derivatives [20 (link)]. Samples were analyzed by gas chromatography (GC; Type: 6890N, Agilent Technologies, Santa Clara, USA) and mass spectroscopy (MS; Type: 5973N, Agilent Technologies). The GC and MS analysis was performed as described in detail previously [21 (link)]. The quantification of the monomers was performed using a gas chromatograph combined with a flame ionization detector. Four replicates of each plant species were used.