Cresol red

For T. palmi, DNA was extracted from individual adults, for B. tabaci it was extracted from larvae and for the fruit flies it was extracted from approximately 1 mm³ of larval tissue. For DNA extraction, tissue samples were added to 30 µl of an alkaline lysis solution [600 µm potassium hydroxide (Sigma‐Aldrich Corp., St Louis, MO, USA) and 2 µm Cresol Red (Sigma‐Aldrich Corp.)] and heated to 95 °C for 5 min on a heat block (Thermomixer Comfort; Eppendorf AG, Hamburg, Germany). The DNA extract was used directly for the LAMP reaction without any purification step.

The entire FCGR3A gene, including the 5′UTR and 3′UTR, was amplified using an FCGR3A gene-specific forward primer and a generic reverse primer, producing a 9654 bases long polymerase chain reaction (PCR) product. The PCR reaction contained 300 ng of genomic DNA, 67 mM Tris-HCl (pH 8.8) (Merck, Darmstadt, Germany), 16.6 mM ammonium sulfate (Merck), 0.01% Tween 20 (Merck), 1.5 mM MgCl₂ (Life Technologies, Austin, Texas), 0.2 mM of each dNTP (GE Healthcare, Diegem, Belgium), 0.1 µg/µl cresol red (Sigma-Aldrich, St. Louis, Missouri), 5% glycerol (Alfa Aesar, Karlsruhe, Germany), 15 pmol of each primer (Sigma-Aldrich) and 2.5 U of Expand Long Template PCR System (Roche, Basel, Switzerland) with a final volume of 30 µl. The PCR program consisted of an initial denaturation step of 2 minutes at 94 °C; followed by 10 cycles of 15 seconds at 94 °C, 30 seconds at 63 °C and 4 minutes at 68 °C; then 10 cycles of 15 seconds at 94 °C, 30 seconds at 60 °C and 6 minutes at 68 °C; afterwards 10 cycles of 15 seconds at 94 °C, 30 seconds at 60 °C and 10 minutes at 68 °C; and a final elongation step of 7 minutes at 68 °C. The PCR products were checked by electrophoresis using a 1.5% agarose gel containing 0.5 µg/µl ethidium bromide (Sigma-Aldrich).

To determine if the strains produce putrescine, strains were grown in Moeller Decarboxylase broth [21 (link)]. Briefly, the broth contained bacteriological peptone (Oxoid) 5 g, meat extract (Merck) 5 g, glucose 0.5 g, bromcresol purple 0.01 g, cresol red 0.005 g, pyridoxal-5’-phosphate 0.005 g (SigmaAldrich), and L-arginine 10 g (SigmaAldrich) per 1 L of medium. The final pH was set to 6.0±0.2, and the medium was autoclaved at 121°C for 15 min. The strains were inoculated in the medium at 1 % (v/v) and incubated at 30°C for 24 h. A yellow colour indicated a negative reaction, and a purple colour indicated a positive reaction (i.e. putrescine production).

Glucose oxidase from Aspergillus Niger (GOx type X-S), horseradish peroxidase, Alkaline phosphatase (Type I-S, bovine intestinal mucosa), Cresol red, Irgacure 819 (phenyl bis(2,4,6-trimethylbenzoyl)-phosphine oxide), poly(ethylene glycol) diacrylate with mean molecular weight of 700 DA (PEG700DA), luminol (3-aminophthalhydrazide), collagen from calf skin type I, Trizma^® base (Tris(hydroxymethyl)aminomethane), calcium chloride, α-d-glucose-1-phosphate (G1P) disodium salt hydrate, silver nitrate and sodium thiosulfate were purchased from Sigma (Lyon, France). Potassium chloride, Veronal (diethylmalonylurea sodium) and sodium phosphate were obtained from Prolabo (Fontenay-sous-Bois, France). Phosphate Buffer Saline (PBS) tablets were purchased from Applichem GmbH (Darmstadt, Germany). All solutions were prepared using milliQ water.

Phenol red (98%, pure), cresol red (98%, pure), p-toluenesulfonyl chloride (99%, pure) and mPEG (polyethylene glycol monomethyl ether, average Mn: 4000 and 5000) were purchased from Sigma-Aldrich Co., LLC (Beijing, China). Atenolol (99%, pure), metoprolol (99%, pure), and phenytoin sodium (99%, pure) were provided by Wuhan Dahua Pharmaceutical Co., LTD (Wuhan, Hubei, China). Ketoprofen (99%, pure) was provided by Hubei Xunda Pharmaceutical Co., LTD (Wuxue, Hubei, China). Ranitidine (99%, pure) was provided by Jiangsu Zhengji Pharmaceutical Co., LTD (Huaian, Jiangsu, China). Ibuprofen (99%, pure) was purchased from Liaoning Pharmaceutical Materials Co., LTD (Shenyang, Liaoning, China). Both solvents of acetonitrile and methanol (Merck KGaA, Darmstadt, Germany) for high performance liquid chromatography (HPLC) analysis were of HPLC grade. All other chemicals were of analytical reagent grade.

Cresol red, bromocresol purple, bromocresol green, and bromothymol blue were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetone and 2-butanone were obtained from Junsei Chemical Co. (Tokyo, Japan), and eco-DEHCH was obtained from Hanwha Solution Corp. (Seoul, Republic of Korea). Industrial filter paper (HC-50), polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF) as gas-permeable membranes were acquired from Hyundai Micro., Ltd. (Seoul, Republic of Korea). Breathron was procured from Nitto Denko Corp. (Osaka, Japan). Cellulose acetate, ammonia solution 30% (w/v), potassium carbonate, anhydrous 99.5% (w/v), sodium hydroxide, 0.01 N H₂SO₄, and 0.01 N NaOH were purchased from Samchun Chemical Co. (Seoul, Republic of Korea). All other chemicals were analytical grade.

pH was measured in the larval midgut using Thymol blue, m-Cresol purple, Cresol red, Bromocreosol purple, BromoThymol blue, Phenol Red and Congo red indicator dyes (all purchased from Sigma- Aldrich). Indicators were added to melted Drosophila diet (0.1% w/v), immediately mixed, and allowed to cool to room temperature. Larvae of the appropriate genotype were added, and after 2 hours the midgut excised in Schneider’s insect medium (Invitrogen). Micrographs were taken immediately using a Sony CyberShot NEX-C3 mounted on a Leica stereo microscope, as pH remains stable for only a few minutes after dissection. Images were processed using Adobe Photoshop CS5.1. Where noted, the diet was also supplemented with 1 mM Omeprazole (Sigma-Alrdich) or 100 μM acetazolamide (Sigma-Aldrich).