Serum biochemistry suggests grey squirrels (Sciurus carolinensis) have poorer physiological condition in urban settings

Human food waste in cities presents urban wildlife with predictable, easily accessible high-calorie food sources, but this can be both beneficial and harmful for individual health. We analyzed body condition and serum chemistry (electrolyte levels, markers of kidney and liver function, protein, glucose, and cholesterol) in an urban and rural population of eastern grey squirrels (Sciurus carolinensis) to assess whether proximity to the human food waste that is associated with urban habitats had ill effects on health. We found no differences in body condition between habitats and no evidence of malnutrition at either site. However, urban squirrels had higher blood glucose, lower potassium, phosphorus, chloride, and albumin:globulin ratios. These results align with previous findings of increased dietary sugar in cities, and suggest that urban populations of grey squirrels are under greater environmental stress than rural populations.


7
Statistical analysis.-Boxplots were created in R version 3.6.1 (33). We first plotted and 128 visually inspected the data, then used principal components analysis (PCA) to visualize 129 groupings, if any. We then tested differences in serum variables between sites using non-130 parametric Kruskal-Wallis tests due to small sample size and unbalanced groups. We found no 131 sex differences for serum measurements (Fig. 1, 2, 3), thus did not include sex as a factor in our 132 analyses.  (Table 1). There were no detectable differences in body condition between 138 sexes (Table 1). 139 Urban squirrels had higher glucose levels than those from the rural site (P <0.01, X 2 = 8.70; Fig.   140 1, Table 1), in line with our expectations. Cholesterol levels did not detectably differ by location 141 (Fig. 1, Table 1). Among serum ions, potassium (P = 0.02, X 2 = 5.57), phosphorus (P < 0.01, X 2 142 = 9.6), and chloride (P = 0.01, X 2 = 5.95) were lower in urban squirrels (Fig. 1, Table 1).

8
The ratio of albumin to globulin (A:G ratio) was higher in rural squirrels (P = 0.05, X 2 = 3.91;

150
Seven principal components had eigenvalues greater than 1-these accounted for 91% of the 151 variation in response variables. PC1 (25.6% of total variation) captured the variation between 152 sites (Fig. 4, Table 2). Chloride, phosphorus, glucose and AST loaded most strongly on PC1 153 ( Table 2). In general, serum parameters at both sites were within published ranges for grey squirrels (23, populations in creatinine and urea, in both cases they tended to be lower in urban populations.

195
Total protein was not different between sites and was on the high margin compared to previously 196 published intervals (23, 34), indicating that neither population was malnourished, at least with 197 respect to protein intake. Serum albumin was comparable in urban and rural squirrels, and 198 though globulin trended higher in urban squirrels, the difference was small. However, we found 199 significantly lower ratios of albumin to globulin (A:G ratios) in urban squirrels. Globulin is   (Figs. 1, 2, 3). We note however, that those ranges were obtained from samples taken year-216 round, and thus might obscure potential seasonal variation in analyte levels. Autumn 217 hyperphagia and rapid weight gain would be expected to elevate serum glucose and lipid markers 218 including cholesterol and albumin, which in mammals is involved in fatty acid transport via the 219 circulatory system (53). Seasonal variation in diet and food intake in grey squirrels (23) would 220 also drive seasonal differences in blood chemistry, making comparison of our values to annual 221 averages in serum parameters not ideal.

223
Our main findings-higher glucose, shifts in electrolyte balance, and lower A:G ratio-suggest 224 physiological responses to differing habitat quality between urban and rural sites. Taken 225 together, this is evidence that urban grey squirrels are less healthy than rural ones in our study 226 populations. We note that this was an exploratory analysis, and results should be interpreted with