Infection
To survey gastrointestinal helminth infection status, the stomach, caecum, small intestines and large intestines were dissected under a microscope and all parasites observed were visually identified and recorded, including both larval and adult forms. Two species of helminth worms were commonly found in the gastrointestinal tracts of the dissected animals: the whipworm Trichuris muris and the pinwormSyphacia obvelata . Also recorded was the tapewormHymenolepsis sp. , though this was rarely recorded (5 instances over 366 recorded mice) and was not included in further analysis.
To measure the level of infection by the hepatic helminth Calodium hepaticum (also known as ­Capillaria hepaticum ), the liver was visually assessed during initial dissection and scored based on the level of decolouration (from 0 = no decolouration, to 5 = extensive discoloration).
To assess ectoparasite infections, following dissection, the fur of each mouse was examined under a dissecting microscope with all parasites identified using a visual key (key created at University of Nottingham, from photos by Laura Myhill), and total number of each species counted. As fleas rapidly move to and from the body, including after capture and after death, numbers are highly dynamic, and therefore only prevalence was recorded.
To record microparasite prevalence, DNA was extracted from the frozen blood clots using a 2-step extraction kit (Extracta DNA Prep, Quanta Bio Massachussetts, USA), and the resulting DNA used for PCR. The microparasites selected for detection were the apicomplexan protozoanBabesia microti (although in fact another closely related apicomplexan was detected instead – see below), the flagellate protozoan Trypanosoma sp. and the bacteria Anaplasma phagocytilum and Bartonella spp. (see Supplementary Materials). These parasites were chosen based on species and genera which are commonly detected across wild rodent populations (Duh, Petrovec, Trilar, & Avsic-Zupanc, 2003; Healing, 1981; Taylor et al., 2018).
Abundance measures of these parasites could not be reliably recorded, and so only presence/absence data from these species are used in analyses. Amplified DNA was run on a 2% agarose gel for 30 minutes at 100V, and visualised under ultraviolet light to confirm amplification. Identity of successfully amplified samples was confirmed through sequencing at an external company (Source Bioscience, Nottingham, UK) and comparison against reference sequences on BLAST (National Center for Biotechnology Information). Successfully amplified samples fromB.microti primers were found to actually be from infections by another apicomplexan parasite, Sarcocystis dispersa . The identity of this parasite was further confirmed through PCR of an additional gene with Sarocycstis -specific primers, sequencing of PCR product as described above.
Infection by C.hepaticum was not recorded for the first 150 mice caught, and blood clot material for these mice was lost, so no C. hepaticum or microparasite data was available for 150 of 366 mice.