Agar powder

Modified Bifidobacterium broth (1000 mL) with pH 6.5 ± 0.2 was prepared using peptone special (22.2 g), NaCl (4.8 g), and L-cysteine hydrochloride monohydrate (0.5 g) with prebiotics and sugars. Four different sugars (fructose, sucrose, glucose, and lactose) at different concentrations (1, 2, 3, and 4%) with FOS supplementation at different concentrations (0.5, 1, 2, 3, and 4%) were used for growth and survival assays. Agar powder, phosphate-buffered saline (PBS) tablets, anaerogens, peptone special, sodium chloride (NaCl), and L-cysteine hydrochloride monohydrate were purchased from Oxoid, UK. Glucose, fructose, sucrose, lactose, and gram staining kit were purchased from Sigma-Aldrich, USA. Fructooligosaccharide (FOS) was obtained from Fiatec Biosystems Sdn. Bhd, Malaysia, with a degree of polymerization between 3 and 8.

A procedure was developed for measurement of the insertion force with and without ultrasound in agarose gel, which serves as a tissue simulant. Agarose (Agar Powder, Acros Organics) gels of various concentrations (weight/volume (w/v)) were prepared by dissolving powdered agarose in distilled water. The solution was sealed and heated for 20 min at 90–95 °C on a hot plate and stirred frequently by hand until dissolved completely. The solution was then poured into Petri dishes to cool at room temperature and cure for at least 6 h. A glass slide was placed on top of the force transducer (Chatillon DGGS) and the agarose gel was placed on top of the glass slide. The ultrasonic probe was mounted on a motorized micromanipulator (MP285, Sutter Instrument). Insertion forces were measured while the probe was inserted into the tissue simulant at various constant velocities. The force transducer and micromanipulator were all affixed on an optical table to minimize the misalignment between the probe tip and tissue stimulant.

DGRP lines were obtained from the Bloomington Stock Center and reared at room temperature on a standard fly medium. The fly medium recipe that we used is the following (for 1 l water): 6.2 g agar powder (ACROS N. 400400050), 58.8 g Farigel wheat (Westhove N. FMZH1), 58.8 g yeast (Springaline BA10), 100 ml grape juice, 4.9 ml propionic acid (Sigma N. P1386), 26.5 ml of methyl 4-hydroxybenzoate (VWR N. ALFAA14289.0) solution (400 g/l) in 95% ethanol and 1 l water. For RNAi (IR) studies, F1 progeny carrying one copy of the da-Gal4 or MyoIA-Gal4 with tub-Gal80^ts transgenes (and Diptericin-lacZ reporter in the case of da-Gal4) as well as one copy of UAS-IR (all in the w¹¹¹⁸ background) were kept at 18 °C for 3 days post eclosion, and then moved to 29 °C for 8 days to activate the UAS-IR. The UAS-Gyc76C-IR line is a gift from Julien Dow, the UAS-Nrk-IR (CG4007 R2 and R3) fly lines were obtained from the DGRC stock centre. Imd pathway mutants used are Dredd^B118 (ref. 30 (link)) and Relish^E20 (ref. 54 (link)).

DGRP lines were obtained from the Bloomington stock center and reared at room temperature on a standard fly medium. The fly medium recipe that we used is the following: 6.2-g Agar powder (ACROS N. 400400050), 58.8-g Farigel wheat (Westhove N. FMZH1), 58.8-g yeast (Springaline BA10), 100-ml grape juice, 4.9-ml Propionic acid (Sigma N. P1386), 26.5 ml of methyl 4-hydroxybenzoate (VWR N. ALFAA14289.0) solution (400 g/l) in 95% ethanol, and 1-L water. We used w¹¹¹⁸ and yw flies as wildtype. The UAS-lark RNAi line was obtained from the Transgenic RNAi Project (TRiP.JF02783), and the UAS-lark-3HA line was obtained from Bloomington stock center (stock # 7125). The P-element insertion lines in lark were obtained from Bloomington stock center (stock #15287 and #22604). Oral infection was performed using a standard protocol as in [13 ]. Survival was counted every 24 h.
For specific knockdown or overexpression of lark in the adult gut enterocyte, F1 lines carrying a copy of the MyoIA-Gal4 and tub-Gal80^ts transgenes [51 (link)], as well as one copy of either the UAS-IR or the UAS-ORF was kept at 18 °C for 3 days post-eclosion, and then moved to 29 °C for 8 days to activate the UAS transgenes. Flies were subsequently infected with P.e. using the standard oral infection protocol (OD₆₀₀ nm of 100 and 1.5% sucrose) [13 ].

DGRP lines were obtained from the Bloomington stock center and reared at room temperature on a standard fly medium with 12-h light dark cycle. The fly medium we used is composed of (for 1 L water): 6.2 g Agar powder (ACROS N. 400,400,050), 58.8 g Farigel wheat (Westhove N. FMZH1), 58.8 g yeast (Springaline BA10), 100 ml grape juice, 4.9 ml Propionic acid (Sigma N. P1386), 26.5 ml of methyl 4-hydroxybenzoate (VWR N. ALFAA14289.0) solution (400 g/l) in 95% ethanol. We used w¹¹¹⁸ and bw;st flies as wildtype. Various DGRP lines, ntc^f03797 and ntc^f07259 stocks were obtained from the Bloomington Stock Center. The bw;st,ntc^ms771/TM6B mutant stock was a kind gift from the Hermann Steller lab.