M3301 micromanipulator

Overnight cultures and extracts of L. pseudomesenteroides were reconstituted in PBS as described above. Aphids were held in place by applying a vacuum and were injected ventrally between the hind legs [19 (link)]. We injected 25 nL of the bacterial suspension or the same quantity of 1× cell-free extract using an M3301 micromanipulator (World Precision Instruments) with glass capillaries. For the live cell injections, the negative controls were mock injections with the same volume of PBS. For the extract injections, the negative controls were mock injections with the same volume of extract prepared from sterile MRS culture medium and reconstituted in PBS. We injected 20 aphids per treatment in three biological replicates. Injected aphids were placed individually for 10 days in Petri dishes containing V. faba leaves on a 1.5% agar substrate [19 (link)] and survival was checked daily. Petri dishes with leaves were replaced after 5 days to ensure the aphids were maintained under optimal conditions.

The double-strand RNA (dsRNA) was synthesized using a Transcript Aid T7 High Yield Transcription Kit (Thermo Scientific, Wilmington, DE, United States) according to the manufacturer’s instructions (Supplementary Table S1). The dsRNA was diluted with Nuclease-free water to a final concentration of 5,000 ng/μL.
Newly emerged green and red form adults (≤12 h old) from the NY strain were used in the RNAi experiments. The dsRNA injection was accomplished as in a previous study (Ye et al., 2019 (link)). Briefly, 600 ng of dsGGPPS and dsGFP (negative control) were injected into the aphids using an M3301 micromanipulator (World Precision Instruments, Sarasota, FL, United States). In each treatment, 25 aphids were injected as one biological replicate and 4 biological replicates were completed. After injection, the aphids were moved to broad bean leaves. After 36 h, in each biological replicate, 5 aphids were collected to detect gene expression levels and 20 aphids were collected for analyzing carotenoids content.

The dsRNA was synthesized using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, Wilmington, MA, United States), according to the manufacturer’s instructions. The injection of dsRNA was performed using a M3301 micromanipulator (World Precision Instruments, Sarasota, FL, United States). Briefly, boiled 1% agarose was poured into a petri dish and allowed to cool until solid. Then, two cross-shaped grooves were made in the agarose for the immobilization of the aphids. Glass capillaries (3.5-in 3-000-203-G/X micropipettes, Drummond Scientific, Broomall, PA, United States) were prepared with a P-97 Micropipette Puller (Sutter Instrument, Novato, CA, United States) using the following parameters: Heat = 575, Pull = 145, VEL = 145, and DEL = 100. During the injection, the aphids were first immobilized by incubating on ice for 5 min, and then placed on the agarose plate before 120 nL of the dsRNA or water (control) was injected laterally, between the middle and hind legs of the adult aphids (Sapountzis et al., 2014 (link)). Each treatment was injected into 25 aphids. Subsequently, the aphids were moved to Vicia faba leaves and kept in petri dishes.