Supplementary Methods
Permits
Experiments were conducted under the following permits: Virginia Tech IACUC (10059-BIOL), Alabama Department of Conservation and Natural Resources (5436), United States
Fish and Wildlife Service (MB158404-1), United States Geological Survey Bird Banding Lab
(23513).
Study Subjects, Housing, and Inoculation
Twenty-eight female, hatch-year house finches were captured on the campus of Auburn
University in Auburn, Alabama, USA (32° 25’ 29.78” N, 85° 29’13.59” W) on 9 and 10
September, 2011. Birds were transported via temperature-controlled state vehicle to Blacksburg,
VA, a trip of roughly 8 hours, on 10 September, 2011. While only females were used in this
study, previous work in this species has shown minimal to no sex effects on pathology or
pathogen load in response to Mycoplasma gallisepticum (MG) infection, and no effects of sex on
responses to novel antigens or on the total amount of food consumed [1-6], suggesting that
results of the current study apply to both sexes. No birds showed clinical signs of MG or had
detectable pathogen load (see methods below) prior to the start of the experiment. One MGtreated bird was excluded from the experiment when it was found to be seropositive for anti-MG
antibodies prior to treatment [7]. One control bird was also found to be seropositive, but was
retained in the analyses.
Birds were housed in pairs in indoor cages (76cm x 46cm x 46cm) for two months at
temperatures ranging between 20-23°C, then moved to individual cages of the same size two
weeks prior to the start of the experiment. Ten days before experimental inoculation, birds were
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placed into one of two temperature-controlled rooms, each with a lighting regime of 11hrs light
and 13 hrs dark. Initial temperatures were a constant 22°C and were gradually adjusted on days 8, -6, and -4 prior to inoculation to yield final thermoneutral temperatures of 28°C during the day
and 22°C at night, and final sub-thermoneutral temperatures of 19°C during the day and 13°C at
night [8-10]. For thermoneutral birds, day-time temperatures were increased by 2°C on each
adjustment day; for sub-thermoneutral birds, day-time temperatures were decreased by 1°C and
night-time temperatures were decreased by 3°C on each of these adjustment days [10].
General Notes on Captive Studies of House Finches
While field studies provide invaluable insights into disease dynamics, and MG in
particular, a full understanding of such systems requires experimental work, which oftentimes
must be conducted in captivity for both ethical and logistical reasons. However, captive studies
of organismal responses to stressors, which can include disease, are justifiably criticised, and
require some evidence that captive animals provide insight into their wild conspecifics [11].
Endocrine data suggest that experimental studies of captive house finches are viable tools
for understanding organismal responses to MG in the wild. First, with acclimation periods to
captivity comparable to or shorter than those in the current study, baseline and stress-induced
corticosterone levels are highly similar between captive and free-living house finches. Both
baseline and stress induced (30min restraint in cloth bags) corticosterone levels were comparable
among free-living house finches in upstate NY [12] (means ± 1SE estimated from figure 3 in
reference [12], baseline: 6.4 ± 1.3 ng/mL; stress-induced:18.9 ± 1.5 ng/mL) and those caught
from the same population at the same time and held in captivity for 2-3 weeks [4] (means ± 1SE
estimated from figure 3b in reference [4], baseline: 4.9 ± 1 ng/mL; stress-induced: 16.9 ± 2.5
2
ng/mL). Additionally, Adelman and Hawley (unpublished data) found that captive house finches
have baseline corticosterone levels comparable to their free-living counter parts [12], both when
healthy and infected with MG (males held in captivity for 3.5 months (n=4), means ± 1SE: PreInfection: 6.5 ± 1.1 ng/mL, Post-Infection: 12.4 ± 2.9 ng/mL; free-living: (values estimated from
figure 3 in reference [12]: Healthy: 7.5 ± 1 ng/mL, Infected: 11 ± 5 ng/mL). Moreover, freeliving birds from AL (location of origin for birds in the current study) show similar baseline
corticosterone levels as do birds from upstate NY, suggesting that comparisons across studies
mentioned here are germane to the present experiment [12, 13].
Inoculation
On day 0, birds were inoculated bilaterally in the palpebral conjunctiva with 40µL of
Mycoplasma gallisepticum suspended in Frey’s media or media alone. We used the 7th in vitro
passage of a MG isolate collected in Virginia, USA, in 1994 (7994-1 7P 2/12/09, David H. Ley,
NC State University, College of Veterinary Medicine, Raleigh, NC, USA 27606), which had a
viable count of 2.24 x 107 colour changing units (CCU), as determined by the most probable
number method [14]. As this strain has been used for experimental infections in numerous
publications, its use facilitates comparison among studies [15]. Moreover, this strain has been
found circulating in wild house finch populations recently, indicating its continued ecological
relevance [16].
Eye Lesion Scoring
Eye lesions were scored on a scale of 0-3 per eye by two observers on each day of
sampling. Briefly, scores corresponded to pathology as follows: 0) no pathology, 1) mild
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swelling around the eye, 2) moderate swelling and mild eversion of the conjunctiva, 3) severe
swelling and substantial discharge. Left and right eye scores were each averaged between
observers and then summed to yield a total eye score, with a highest possible value of 6 [7] .
Food Consumption Details
Food consumption was calculated as:
𝑓𝑜𝑜𝑑 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 = 𝑓𝑒𝑒𝑑𝑒𝑟 𝑚𝑎𝑠𝑠𝑖𝑛𝑖𝑡𝑖𝑎𝑙 – (𝑓𝑒𝑒𝑑𝑒𝑟 𝑚𝑎𝑠𝑠𝑓𝑖𝑛𝑎𝑙 + 𝑠𝑝𝑖𝑙𝑙𝑒𝑑 𝑓𝑜𝑜𝑑 𝑚𝑎𝑠𝑠)
Foraging Behaviours
During focal behavioural sampling of video, the number of times each bird pecked at the
feeder port and the amount of time each bird spent within one body-width of the feeder were
quantified using JWatcher [17]. Periods of time in which birds were immobile (no detectable
movement), but within one body-width of the feeder were not counted as time at the feeder, as
this inactivity precluded any feeding behaviour. Including these periods of inactivity did not
qualitatively alter our results.
Additional Statistical Details
Linear mixed effects models were performed on conjunctival pathogen load, conjunctival
lesions, food consumption, and foraging behaviours in R [18, 19]. Residuals from all models
followed normal distributions, suggesting that assumptions of the techniques were met.
Because our two measures of foraging behaviour, number of pecks at food and time spent
within one body-width of the feeder, were significantly correlated (Spearman’s rho = 0.59, p <
0.001), we performed a principal components analysis to collapses these metrics into a single
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variable to be used in analysis of pathogen deposition. Both behavioural metrics loaded with the
same sign onto PC1, which explained 76% of the variation in these two variables (table S2). As
such, we used PC1 as our metric of foraging behaviour in the additive mixed model explained
below.
Because time series data are often autocorrelated, mixed effects models of all variables
included a function adjusting for temporal autocorrelation if this function improved overall fit of
the maximal model, as assessed by AICc [20]. Autocorrelation functions tested were those
included in the “nlme” package for R [19].
Because we were interested in the amount of pathogen from the conjunctiva that a bird
deposited on a feeder, we assessed the effect of ambient temperature on pathogen deposition
while controlling for individual conjunctival pathogen load, mass, and foraging behaviours using
a generalized additive mixed effects model (GAMM). The initial model contained fixed effects
of temperature treatment, mass (taken 2-4d prior to other metrics), PC1 (see above), and separate
functions of pathogen deposition by conjunctival pathogen load (cubic spline smoothers) within
each temperature treatment, incorporating bird ID as a random effect [21]. We simplified this
model by first using only a single function of deposition by conjunctival load for all birds, then
by removing the main effects of other variables, beginning with the highest p-values. The model
fits were assessed using ΔAICc values; models with ΔAICc values < 2 were presumed to be
significantly better fits to the data [20]. To determine how conjunctival pathology and food
consumption affected pathogen deposition at the individual level, while controlling for all
variables in the initial, maximal GAMM, we performed a linear mixed effects model on the
residuals from that model, using total eye score and food consumption as main effects and bird
ID as a random effect.
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Effects of Temperature on MG Detection
To determine if ambient temperature influenced our ability to detect MG DNA on inert
substrates (e.g. bird feeders), we performed an in vitro incubation experiment. MG stock (isolate
2006.080-5-4P, (~2.5 X105 CCU/ml, David Ley, North Carolina State University) was serially
diluted to yield four working solutions: full strength, 1:10 dilution, 1:100 dilution, and 1:1,000
dilution. We placed 5μL of each solution onto each of six replicate glass microscope slides.
Three of the replicates from each dilution were incubated at 19oC, the other three at 28oC (the
daytime temperatures for the sub-thermoneutral and thermoneutral groups of the main
experiment, respectively). After nine hours (equivalent to the time between lights-on and feeder
swabbing in the main experiment), slides were swabbed for 10s with a sterile cotton swab dipped
in TPB, in the same manner that feeders were swabbed the main experiment. Swabs were then
swirled and wrung out into a tube containing 100 l of TPB solution and stored at -20oC until
DNA extraction the following day. DNA extraction and qPCR were performed as in the main
methods section [7, 22]. Data were analyzed using ANOVA, with dilution, incubation
temperature, and their interaction as main effects.
Neither incubation temperature (F1,16 = 0.15, p = 0.71) nor the interaction between
incubation temperature and dilution (F3,16 = 0.07, p = 0.75) influenced the amount of MG DNA
detected (Figure S1). This result suggests that the observations of MG deposition in our main
experiment were not biased by any effect of temperature on MG detection probability.
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Supplementary Tables and Figures
Table S1. Timeline of sampling during the experiment.
MG load MG deposition
food
week
day
in eyes
on feeders
consumed
0
0
inoculation
1
4
X
X
X
2
12
X
X
X
3
17
X
X
X
4
24/25
X
X
X
(MG = Mycoplasma gallisepticum, X = sampling performed)
Table S2. Summary of the principal components analysis on food consumption and feeding
behaviours.
variable
number of pecks at food
proportion of time within one
body-width of feeder
cumulative proportion of
variance explained
rotation PC1
-0.71
rotation PC2
0.71
-0.71
-0.71
0.76
1.00
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Figure S1. Ambient temperature did not influence our ability to detect Mycoplasma
gallisepticum (MG) on glass slides using quantitative PCR. 5μL aliquots of four different
dilutions of MG were incubated on glass slides for nine hours at either 19°C or 28°C. Bars show
means ± 1SE for three replicate slides at each combination of temperature and dilution.
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