Experiment 1 – Diet manipulation
The average amount of food that birds consumed did not vary by diet
treatment (χ2 = 1.75, P = 0.416). Body mass
decreased over the course of the experiment (day: χ2 =
49.49, P < 0.0001), but did not vary by treatment
(Figure S1; χ2 = 0.36, P = 0.836) or the
interaction between treatment and time (χ2 = 0.38,P = 0.829). We observed a non-significant trend where fat score
also decreased over the course of the experiment (Figure S2; day:
χ2 = 3.61, P = 0.058). Fat score did not vary
by treatment (χ2 = 0.47, P = 0.792) or the
interaction between treatment and time (χ2 = 1.29P = 0.526). Corticosterone concentrations significantly increased
after 30 min, being higher in stress-induced samples (Figure S3; sample
type: χ2 = 150.99, P < 0.0001), but
were not affected by diet treatment (P = 0.124) or time sampled
(P = 0.060). Complement activity was also unaffected by
treatment, day, and the interaction between treatment and day (Figure
S4; all P
Bacterial richness and bacterial diversity of the gut microbiota did not
vary across diet treatments, over time, or in response to the
interaction between treatment and time (Table S1; Figure S5; allP adj ). However, when considering the interactive
effects of each physiological metric (Table S2; complement, baseline
corticosterone, stress-induced corticosterone) with diet treatment and
time (day 0, day 8), we found that complement activity and changes in
complement activity over time significantly affected microbial richness
and Shannon diversity. Higher complement activity levels in blood plasma
were associated with greater richness (Figure 2; complement:P adj = 0.0280) and higher Shannon diversity
(Figure 2; complement: P adj = 0.0492). This
relationship was strongest prior to treatment (day 0), and appeared to
be disrupted over the course of the experiment (complement*day;
Richness: P adj = 0.0023, Shannon diversity:P adj = 0.0118). Shannon diversity was also
influenced by a significant three way interaction between treatment,
time, and stress-induced corticosterone concentrations (Figure 3;
treatment*day*SIcort: P adj = 0.0276). Higher
stress-induced plasma corticosterone concentrations were generally
associated with higher Shannon diversity, however this positive
association was not apparent in birds on a protein diet prior to
treatment (day 0) or birds fed a high fat diet for 8 days. We did not
find a significant effect of diet, time, or the interaction between diet
and time on any beta diversity metrics (Table S3, Figure S6; allP adj d that birds on the high protein diet had a
significant reduction in the abundance of the phylum Campylobacterota
(Figure 4; χ2 = 6.75, P adj = 0.0469) when
comparing samples collected on day 0 (pre-treatment) to samples
collected on day 8 (post-treatment). Conversely, there were no
significant changes in relative abundance at the phylum level in the
high fat (all P adj ≥ 0.870) or equal ratio (allP adj ≥ 0.603) diet treatments when comparing pre-
and post- treatment samples (Table 1). We did not detect any significant
changes in any of the three diet treatment groups at the genus level
when comparing pre- and post- treatment samples (Table 1, Figure S7; allP adj ≥ 0.143).