Host-associated bacterial microbiomes can facilitate host acclimation to seasonal environmental change and are hypothesized to help hosts cope with recent anthropogenic environmental perturbations (e.g., landscape modification). However, it is unclear how recurrent and recent forms of environmental change interact to shape variation in the microbiome. The majority of wildlife microbiome research occurs within a single seasonal context. Meanwhile, the few studies of seasonal variation in the microbiome often restrict focus to a single environmental context. By sampling urban and exurban eastern grey squirrel populations in the spring, summer, autumn, and winter, we explored whether seasonal rhythms in the grey squirrel gut microbiome differed across environments using a 16S amplicon sequencing approach. Differences in the microbiome between urban and exurban squirrels persisted across most of the year, which we hypothesize is linked to anthropogenic food consumption; but we also observed similarities in the urban and exurban grey squirrel microbiome during the autumn, which we attribute to engrained seed caching instincts in preparation for the winter. Host behaviour and diet selection may therefore be capable of maintaining similarities in microbiome structure between disparate environments. However, the depletion of an obligate host mucin glycan specialist (Akkermansia) during the winter in both urban and exurban squirrels was among the strongest differential abundance patterns we observed. In summary, urban grey squirrels showed different seasonal patterns in their microbiome than squirrels from exurban forests, however, in some instances host behaviour and physiological responses might be capable of maintaining similar microbiome responses across seasons. 

Sampling Protocol

Adult eastern grey squirrels were trapped from 2016–2018 (Animal Utilization Protocol no. 3506, Wildlife Scientific Collectors Authorization no. 1087323). The main campus of the University of Guelph (43°31052.3300 N, 80°13036.8000 W, Guelph, Ontario, Canada) was used as our urban site, while the deciduous forests of the ‘rare Charitable Research Reserve’ (43°22052.1700 N, 80°20052.4600 W, Cambridge, Ontario, Canada) served as our exurban site. Initial site selection involved qualitative assessments sites (e.g., prominence old growth forest versus human-built impervious surfaces, presence of refuse bins, human foot-traffic); however, post hoc  quantification of normalized difference vegetation index (NDVI) and normalized difference built-up index (NDBI) estimates (surrounding locations of sample collection) demonstrate clear differences in both the amount of vegetated area (NDVI_exurban = 0.426 ± 0.074; NDVI_urban = 0.267 ± 0.081) and the footprint of human-built constructs (NDBI_exurban = -0.311 ± 0.024; NDBI_exurban = -0.165 ± 0.108) between sites (Rimbach et al., 2022). Both study sites reside on the treaty lands and territory of the Neutral, Haudenosaunee, and the Anishinaabe peoples.

Squirrels were captured using tomahawk Model 102 traps (Tomahawk Live Trap Co., WI, USA). Traps were baited with either peanuts or oat and peanut butter balls and checked every hour from 0600–1600. Following capture, squirrels were transferred to a cloth bag until fecal pellets could be collected (approximately 15 minutes). Sex and reproductive condition were recorded at the time of capture. Reproductive condition was coded as either reproductive female (lactating or in estrus), non-reproductive female, scrotal male, or non-scrotal male. All squirrels were provided with a unique pair of alpha-numeric ear tags for future recapture identification. Fecal pellets were stored on ice in the field until transfer to storage at -20 ºC. In total, we collected and sequenced fecal samples from 112 unique individuals, to avoid pseudo-replication at the individual-level. We collected a total of 64 samples from urban squirrels (n_spring: 20, n_summer: 17, n_autumn: 10, n_winter: 17), 27 of which were from males (n_scrotal = 21; n_non-scrotal = 6) and 37 were from females (n_reproductive = 7; n_{non-reproductive} = 30). At our exurban site, we collected a total of 48 samples (n_spring: 15, n_summer: 20, n_autumn: 5, n_winter: 8), 27 of which were from males (n_scrotal = 22; n_non-scrotal = 5) and 21 were from females (n_reproductive = 3; n_{non-reproductive} = 18). 

Sequencing and Bioinformatics

We extracted DNA from 0.2 g of feces using QIAamp DNA Stool Mini Kits (Qiagen, Hilden Germany). The v4 region of the 16S rRNA gene was amplified in triplicate (primers, 515F: 5’-GTGYCAGCMGCCGCGGTAA-3’; 806R: 5’-GGACTACNVGGGTWTCTAAT-3’) at MetaGenomBio Inc. (Waterloo, Canada; Walters et al., 2015). Triplicate PCR products were pooled and sequenced to a depth of 30,000 reads/sample on an Illumina MiSeq platform (v2 chemistry). Briefly, we merged paired-end reads and performed quality filtering using a standardized mothur pipeline (Kozich et al., 2013). Paired-end reads were clustered to operational taxonomic units (OTUs) based on a 97% 16S rRNA gene sequence similarity using the OptiClust algorithm (Westcott & Schloss, 2017).  Taxonomy was assigned to representative v4 sequences using the SILVA v138 reference database (Yilmaz et al., 2014). Only OTUs which were present at a relative abundance of 0.001 in at least a single sample were retained for analysis to remove singletons and potential sequencing errors. OTU counts were rarefied to the lowest read-count in the dataset (3326 reads) prior to all analyses.

References

Rimbach, R., Grant, A., Gupte, P. R., Newman, A., Stothart, M. R., & Pontzer, H. (2022). Comfortably numb? Regional differences in the relationship between indices of urbanization and a stress indicator in eastern gray squirrels. Urban Naturalist, 9(54), 1–15. http://www.eaglehill.us/urna.

Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K., & Schloss, P. D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the miseq illumina sequencing platform. Applied and Environmental Microbiology, 79(17), 5112–5120. https://doi.org/10.1128/AEM.01043-13

Walters, W., Hyde, E. R., Berg-lyons, D., Ackermann, G., Humphrey, G., Parada, A., Gilbert, J. a, & Jansson, J. K. (2015). Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems, 1(1), 1–10. https://doi.org/10.1128/mSystems.00009-15.Editor

Westcott, S. L., & Schloss, P. D. (2017). OptiClust, an improved method for assigning amplicon-based sequence data to operational taxonomic units. mSphere, 2(2), 1–11. https://doi.org/10.1128/mspheredirect.00073-17

Yilmaz, P., Parfrey, L. W., Yarza, P., Gerken, J., Pruesse, E., Quast, C., Schweer, T., Peplies, J., Ludwig, W., & Glöckner, F. O. (2014). The SILVA and ‘‘All-species Living Tree Project (LTP)’’ taxonomic frameworks. Nucleic Acid Research, 42, 643–648. https://doi.org/10.1093/nar/gkt1209