Whatman filter paper

A virus MC was constructed using virus stocks consisting of tailed phages T5, Det7, and P22, the filamentous phage M13, and the mammalian viruses MHV-68, BVDV-1 strain Ky1203nc [34 (link)], and RV-A strain simian rotavirus SA/11 (Table 1). Virus titres were determined by epifluorescence microscopy using a protocol adapted from [35 (link),36 (link)]. Briefly, a 13 mm Al2O3 Anodisc 0.02 μm filter membrane (Cytiva, Little Chalfont, UK, ref 6809-7003) was placed in a Swinnex filter holder (Millipore, Darmstadt, Germany, ref SX0001300) fitted onto a glass tube protruding through a rubber stopper into a Büchner flask. The flask was connected to a Millivac Maxi vacuum pump (Millipore, Darmstadt, Germany, ref SD1P014M04). After the pump was switched on, 400 μL deionised water or PBS was added to the filter holder to confirm the inlet was sealed. The stained sample was added gradually using a 1 mL micropipette. The filter was then washed with 400 μL of deionised water or PBS and any remaining liquid was aspirated for one minute. The filter was then placed sample-side-up onto Whatman filter paper (Whatman, Marlborough, MA, USA, ref 1004-055) to dry for 5 min. A 7.5 μL drop of Fluoromount G (Invitrogen, Waltham, MA, USA, ref 00-4958-02) was placed on a glass microscopy slide (VWR, Leicestershire, UK, ref 631-0117) and the filter placed on top with 7.5 μL of Fluoromount G added to the filter and covered with a glass cover slip (VWR, ref 631-0125). The slide was then left in the dark at 21 °C for at least 2 h to allow the mountant to set. The slides were imaged on an Axio Imager.M2 upright fluorescence microscope (Zeiss, Cambourne, UK). Samples were illuminated using a HAL 100 illuminator with a quartz collector 43 (Zeiss, Cambourne, UK, ref 423000-9901-000) and a 65HE Alexa 488 filter. For titre measurements, a 100X EC Plan-Neofluar oil immersion objective (Zeiss, Cambourne, UK, ref 420496-990-000) was used. Images were captured on an ICX 285 CCD monochrome camera (Sony, Surrey, UK). Between 15 and 20 images were taken of each filter at random locations.
To prepare the MC, each virus stock was diluted in PBS to obtain the same final concentration, except for MHV-68, for which the concentration was 25% of the concentration of the other viruses. A high concentration mock community (HI) was constructed such that 35 μL of the final MC contained 0.25 × 10⁷ particles of MHV-68 and 1 × 10⁷ of each of the other viruses, for a total of 6.25 × 10⁷ virus particles. A lower amount of MHV-68 was used due to limited availability of stock. As the estimated number of virus particles in human faeces is up to ~10⁹ particles/g [36 (link)], the total number of virus particles in 35 µL HI roughly corresponded to the maximum expected number of virus particles in 50 mg faeces, for a final concentration of 1.25 × 10⁹ MC particles per gram of faeces. A low concentration MC (LO) was produced by 100-fold dilution of HI in PBS, with 35 µL of LO added to a 50 mg faecal sample corresponding to 1.25 × 10⁷ MC particles per gram of faeces. MCs were stored at 4 °C.

The HPLC system (Waters, Milford, MA, USA) consisted of a waters 1525 binary HPLC pump, a Waters 2998 Photodiode Array Detector, and a Waters 2707 Autosampler. The data were acquired and processed using Windows XP-based Waters Breeze 2 software. Ultrapure water (18 MΩ/cm) was produced by a Milli-Q^® Advantage A10^® Water Purification System (Billerica, MA, USA).
The chromatographic separations were carried out on a reverse phase Waters Symmetry^®C₁₈ column (150 × 4.5 mm i.d., particle size 3.5 μm). The mobile phase was a mixture of acetonitrile and 10 mM potassium dihydrogen orthophosphate (65:35, v/v; pH adjusted to 7 with sodium hydroxide) delivered at a flow rate of 1 ml/min. The mobile phase was filtered through 0.45-µm Whatman^®filterpaper and sonicated for 20 min. Analysis was performed at ambient temperature, and the elution of the compounds was monitored by diode array detection (DAD) from 190 to 400 nm. The chromatograms were recorded at 254 nm, and the injection volume was 20 µl.

PER assays were carried out as described in Slone et al. (Slone et al., 2007 (link)) with some modifications. Briefly, files were collected on the day of eclosion and kept in standard corn meal food for 3–7 days at 25°C. Before performing PER assays, flies were starved for 24 to 26 hr at 25°C in vials with a water-saturated Whatman filter paper. Flies were immobilized by cooling on ice, mounted by their backs/wings on a microscope slide using double-sided Scotch tape and allowed to recover for 30 min at room temperature. Prior to the PER assay, flies were allowed to drink water until satiation to ensure PER responses were nutrient derived. Flies that showed no response to water were excluded. Taste solutions were delivered with a 10 μl pipette to legs for up to ~four s. Each fly was tested three times with each taste solutions, and flies were allowed to drink water between each new application. A PER response was recorded as positive (1) if the proboscis was fully extended, otherwise it was recorded as negative (0). PER response scores (%) from a single fly was 0% (0/3 responses in the three applications), 33% (1/3), 66% (2/3) or 100% (3/3).