Laboratory-housed rodent sample collection
Test subjects were 10 male and 10 female bank voles (Myodes glareolus), 8 male and 10 female field voles (Microtus agrestis), 6 male and 10 female wood mice (Apodemus sylvaticus), 10 male and 10 female wild-stock house mice (Mus musculus domesticus) and 10 male and 10 female laboratory rats (Rattus norvegicus). Bank voles and field voles were a mixture of animals wild-caught in Northwest England 9 to 15 months prior to the start of the study and first-generation offspring of individuals wild-caught in Northwest England aged 5 to 18 months. Wood mice were all wild-caught in Northwest England 22 to 27 months prior to the start of the study. House mice were 9 to 17 months old, captive-bred for 5–10 generations from populations captured in Northwest England. Inbred or random-bred laboratory strains were Hsd:ICR (CD-1®, ‘ICR’), C57BL/6JOlaHsd (C57BL/6) and BALB/cOlaHsd (BALB/c) were in-house bred. Rats were 8 to 9 months old from a random-bred cross between Wistar (HsdHan®:WIST, InVivo, Bicester, UK) and Brown Norway (BN/SsNOlaHsd, InVivo, UK) laboratory strains, originally obtained from Envigo UK and subsequently crossbred in-house for three generations. Bank voles, field voles and male house mice were housed singly in 48 × 15 × 13 cm cages (M3, North Kent Plastics, UK). Female house mice were housed in groups of 2 to 4 full siblings in 45 × 28 × 13 cm cages (MB1, North Kent Plastics, UK). Wood mice were housed singly in 38 × 25 × 18 cm cages (RM2, North Kent Plastics, UK). Rats were housed in same-sex pairs in 56 × 38 × 22 cm cages (RC2R, North Kent Plastics, UK).
All animals were fed 5FL2 EURodent Diet (IPS Product Supplies Limited, London, UK) ad libitum and had access to water ad libitum. Wood mice, bank voles and field voles were supplemented with Harry Hamster complete muesli (Supreme Petfoods Ltd., Ipswich, UK) and hay. Field voles were also given fresh-cut grass. All cages had Corn Cob Absorb 10/14 substrate (IPS Product Supplies Limited, London, UK) lining the base. Cardboard tubes and paper wool nest material were provided to all animals for enrichment, with 15 × 8 cm plastic tubes also provided for rats. Animal numbers are summarised in Additional file 6: Table S1.
Collection of faecal pellets
Laboratory-housed rodents were placed individually into a clean laboratory cage for 1 to 2 h, and multiple faecal pellets were collected from each individual. The order of sample collection from each animal was randomised.
Wild rodent sample collection
Longworth traps, Mk1 or Mk2 TubeTraps (BioEcoSS Ltd., Shropshire, UK) and Ugglan traps were set in five separate locations: Kielder Forest (Northumberland, UK); Ness Botanic Gardens (Wirral, UK); Wood Park Farm (Wirral, UK); University of Liverpool, Leahurst Campus (Wirral, UK); and a private garden in Mouldsworth (Cheshire, UK); locations are recorded in Additional file 6: Table S1. Traps, cleaned before every use, were baited with parakeet seed mix (Rob Harvey, Tongham, UK) and a piece of apple. Hay was provided in the traps as bedding material. Traps were checked twice daily. Species and sex of all trapped animals were recorded, and multiple faecal pellets were taken from each trap. Sex was determined using anogenital distance. No faecal samples were taken when more than one animal was captured in the same trap, and animals were fur clipped to avoid repeated sampling. Faecal samples from wild Norway rats (Rattus norvegicus) were obtained as loose droppings from building floors at Wood Park Farm (Wirral, UK), Ness Heath Farm (Wirral, UK) and Shotton Industrial Estate (Wirral, UK). The distance between the sample locations and local population density ensured that samples were very likely to be from different individuals. For the discrimination study for wild rodents, samples were collected from 80 bank voles, 40 field voles, 74 wood mice and 29 rats (Additional file 6: Table S1) and stored for up to 15 days at − 20 °C before the analysis.
Evaluation of storage conditions
Faecal donors were 12 male captive-bred house mice (bred for 5–10 generations from populations captured in Northwest England) aged 11 to 13 months. Multiple faecal samples were collected from each donor and stored in closed Eppendorf tubes at 4 temperatures: − 18 °C, − 4 °C, 21 °C and ambient (mean 18.25 °C, maximum 24 °C, minimum 17 °C). Ambient temperature samples were stored in open or closed Eppendorf tubes. Samples were stored for 1 day, 1 week or 4 weeks. Samples were randomly allocated to each temperature and time condition. For each donor, a sample was stored for each temperature and time condition, giving 15 samples per donor.
Diet study sample collection
Test subjects were 48 singly housed wild-stock male house mice (9 to 18 months old, bred for 5–10 generations from populations captured in Northwest England). All subjects were fed 5FL2 EURodent Diet ad libitum prior to the start of the study. At the start of the study, mice were assigned to four treatment groups (n = 12) and fed different diets. During an acclimation week, subjects were fed a mixture of 5FL2 EURodent Diet and their new diet. From the second week, house mice were fed only their new diet for a further 4 weeks. Diets were Poultry Grower (SDS, Braintree, UK), Harry Hamster complete muesli (Supreme Petfoods Ltd., Ipswich, UK), Turbo 40 pig feed (Massey Bros Feeds Ltd., Crewe, UK) and 5FL2 EURodent Diet (IPS Product Supplies Limited, London, UK). Faecal samples were collected from each mouse on the first day of the study and at weekly intervals over the study.
Sex, maturity and strain study
Test subjects came from two inbred laboratory mouse strains, C57BL/6JOlaHsd (C57BL/6) and BALB/cOlaHsd (BALB/c), and one random-bred laboratory strain, Hsd:ICR (CD-1®, ‘ICR (CD-1)’). The strains were originally obtained from Envigo UK and subsequently bred in-house. They were maintained in MB1 cages, and faecal samples were collected by temporary transfer to M3 cages. All animals were fed on 5FL2 EURodent Diet and had access to water ad libitum. All cages contained Corn Cob Absorb 10/14 substrate, 15 × 5 cm plastic tubes and paper wool nest material. Samples were collected from 176 individuals (details in Additional file 6, Table S1; BALB/c and BALB.K were combined for this study). Samples were stored at 4 °C for up to 7 days or at − 18 °C for up to 30 days prior to analysis.
REIMS processing of faecal samples
All sampling was conducted in a Ductless Fume box (Air Science, Liverpool, UK). REIMS requires that samples contain sufficient water to conduct an electric current to heat the sample and generate fumes. As faecal pellets collected in the field may have dried to a variable degree, we optimised a rehydration protocol. Faecal pellets were placed onto 25-mm glass microfiber filter paper disc (GE Healthcare/Whatman), moistened with MilliQ water. The pellets were then individually hydrated with 200 μL of MilliQ water for 1 to 2 min. An aerosol was generated using a monopolar electrosurgical pencil in either cut mode at 35 W (species discrimination) or coagulate mode at 40 W (mouse age, sex, strain) powered by a VIO 50 C electrosurgical generator. Sampling was of three to five pellets from the same individual and/or condition for data acquisition for 2–5 s per pellet. Sample processing was conducted blind to the treatment condition of the sample, and the order of sample processing was randomised.
Aerosol particles were aspirated using a Venturi gas jet pump powered by nitrogen on the REIMS source via a 3-m evacuation tubing incorporated into the electrosurgical pencil. The Venturi pump introduces the aerosol orthogonally to the inlet capillary of the mass spectrometer, which is then drawn into the source by the vacuum of the instrument. This geometry, combined with a specially designed whistle within the Venturi housing, ensures that the larger particles are not drawn into the capillary where they could cause a blockage. A solution of leu-enkephalin (1.72 pmol/μL dissolved in propan-2-ol) (Fisher Scientific) was infused at 100 μL/min and nebulised at a position opposite the inlet capillary within the whistle assembly. This peptide was used as a lock mass (544.26 m/z) to maintain an accurate mass measurement during all analyses. Laboratory animal and storage studies were conducted using the beta version of the impactor (ceramic cylinder) whereas the wild animal and diet study samples were analysed using the commercial version (Kanthal metal coil). Mass spectra were recorded on a Synapt G2-Si (Waters, Wilmslow, UK) in full-scan resolution, negative ion mode at a scan rate of 1 scan per second from 50–1200 m/z. The sample cone was set to 60 V, and the heater bias was set to 60 V.
An overview of the data analysis workflow is presented in Additional file 2: Figure S1. Individual burn spectra for each faecal pellet were aggregated to generate a single raw data file for each sample. Mass spectra were imported into Waters Offline Model Builder software (OMB-1.1.28, Waters Research Centre, Hungary) or LiveID (Waters). Within Offline Model Builder spectral data above, the intensity threshold of 3 × 105 counts were summed for each data point, accumulating data from multiple faecal pellets from the same animal. Within LiveID, intensity threshold was set automatically, and the exported, binned data were further processed in R. Mass spectra were then lockmass corrected to either a propan-2-ol background peak at 325.19 m/z or leu-enkephalin at 554.26 m/z. For analysis, a mass range of 400 to 1100 m/z was used. The resulting spectra were normalised, scaled and binned by either LiveID or Offline Model Builder (Waters) at 0.05 or 0.1 m/z bin width. Binned data (approx. 14,000 or 7000 data points) were exported as .csv data files for further analysis. For some experiments, data were analysed by principal component analysis (PCA) followed by discriminant function analysis (DFA) using either SSPS version 24 (IBM, Portsmouth, UK) or R. Random forest classification was achieved with package ‘randomForest’  using R version 3.4.2. . For random forest analysis, two analyses were completed—in the first, all samples were included in the classification, and in the second, we retained 70% as a training set and used the trees generated therefore to assess the remaining 30%. A confusion matrix was generated to determine the accuracy of classification for each species or another category. Specific ions that made the greatest contribution to classification were identified using the randomForestExplainer package  in R. Data were visualised with SPSS or with ‘ggplot2’ in the R environment . In some instances, the top informative ions included both the monoisotopic ion and the first 13C isotopomer that were identified and confirmed by plotting a cross-correlation matrix for the intensities of these ions—such isotopically linked pairs exhibited a very high degree of correlation, as would be expected (Additional file 4: Figure S3).