Im 9b microinjector

Double-stranded RNA (ds-RNA) was synthesized following the manufacturer's instructions. In brief, PCR products (for primer pairs see Supplementary Table S4) were amplified with T7 promoter conjugated primer and then purified using the Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI, United States) as templates for in vitro transcription. ds-RNA was synthesized with T7 RiboMAX^TM Express RNAi System (Promega), diluted to 1,000 ng/μL with ddH₂O, and stored at −20°C. Target ds-RNA (10 μg) was delivered into each locust dorsal vessel through the inter-segmental membrane of nymphs on the first day of the fifth instar, using an IM-9B microinjector (Narishige, Tokyo, Japan) equipped with a glass capillary. ds-green fluorescent protein (GFP) was microinjected as a control group. The treated locusts were raised normally like wild-type insects. RNA silencing efficiency was checked on post-injection day 3. The number of biological replicates was at least 4, and the sample number in each replicate was at least 2. All RNAi-treated insects that were used for single sensillum recordings (SSRs) were also checked after recording to confirm the silencing efficiency.

Double-stranded RNA (dsRNA) was synthesized based on the manufacturer’s manual. In brief, PCR products were amplified with T7 promoter-conjugated primers (Supplementary Table S2) and then purified with Wizard® SV Gel and PCR Clean-Up System (Promega, United States) as templates for in vitro transcription. DsRNA was synthesized with the T7 RiboMAX™ Express RNAi System (Promega, United States), and its concentration was determined with an ND-2000 spectrophotometer. DsRNA was then diluted to 2000 ng/μl with ddH₂O and stored at −20°C. 5 µg of dsRNA was injected into each locust’s dorsal vessel through the abdomen’s intersegmental membrane (1st day of 5^th instar nymphs) by using an IM-9B microinjector (Narishige, Japan) equipped with a glass capillary. DsGFP was microinjected as a control group. The treated locusts were raised normally, similar to wild-type locusts. RNAi-treated animals were used for behavioral or electrophysiological experiments on the 3rd day post-injection. After these experiments, intact maxillary palps were isolated and immediately frozen in liquid nitrogen or kept at −80°C. Total RNAs were then extracted using TRIzol reagent. The silencing efficiency of RNAi was checked by RT–qPCR.

The number of microcapsules being injected was one million per animal. The 2 μL suspension of microcapsules in the isotonic solution (0.9% aqueous solution of NaCl) was injected into the central hemolymph vessel of amphipods between the sixth and seventh segments of pereon using the IM-9B microinjector (Narishige, Tokyo, Japan). The injected microcapsules contained FITC-albumin, unless otherwise specified. During the injection, the individual was immobilized inside a wet polyurethane sponge with a temperature of approximately 6 °C.