Msa3.6p

The crude mixture of pigment extracts was used for thin-layer chromatography (TLC). The protocol of TLC was modified from Tsuchida et al. (2010) (link). Based on the values for the G channel (as recorded using a digital camera), the aphids were categorized to have two colors: the red (G value: around 160) and the pale red (G value: around 190). The aphids were quick-frozen in liquid nitrogen and freeze-dried for 36 h in a lyophilizer (MSA3.6P, Sartorius, Germany). They were extracted using a chloroform-ethyl alcohol (1:2) mixture. The total solution was kept in the dark for 24 h and centrifuged at 20,000 × g for 15 min at 4°C to remove deposition from the mixture. The clarified liquid was used for TLC.

Twelve-hour-old fourth instar nymphs of the wingless red and green A. pisum were transferred into transparent plastic dishes (90 mm in diameter; 20 aphids per dish) for starvation treatment. The aphids were collected at hourly intervals for 24 h. The collected samples were then immediately used in energy reserve assays. The aphids were freeze-dried for 36 h in a lyophilizer (Heto PowerDry LL3000 Freeze Dryer, Thermo Fisher Scientific, United States); 2 mg of dried samples was weighed out using a high-precision electronic balance (MSA3.6P, Sartorius, Germany) at ambient temperature. The aphids were transferred into a 1.5-mL microtube, homogenized by a micropestle, and dissolved in 800 μL of solution buffer (100 mM KH₂PO₄, 1 mM DTT, 1 mM EDTA, pH 7.4) for further analysis. This experimental step had three replicates.

Leaves of V. faba, T. repens, C. annuum, N. tabacum and T. aestivum were freeze-dried for 36 hours in lyophilizer (MSA3.6P, Sartorius, Germany). The samples were then moved into a drying oven (Thermo, USA) and dried for 2 hours at 60 °C. After that, 10 mg of treated samples were weighed precisely by a high precision electronic balance (LL3000, Thermo, USA) at room temperature and transferred into 50 ml plastic tubes and covered. All organized samples were then used for L-DOPA and dopamine extraction and assay.
To analyze relative L-DOPA concentrations in plants phloem, 100 g of leaves of V. faba, T. repens, C. annuum, N. tabacum and T. aestivum were cut into 5 pieces and extracted in 1 ml EDTA solution (5 mM, pH = 7.0) for 24 hours (20 ± 1 °C). Extracted solutions were then filtered into a new tube; and 100 mg leaves of V. faba were extracted by grinding as a positive control47 48 (link)49 (link). Each treatment was replicated six times.