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THE WILSON JOURNAL OF ORNITHOLOGY . Vol 123. No. 4. December 2011 
ability of Tree Swallow whole blood to kill E. coli 
would increase over the nestling period in 
synchrony with physical growth and would reach 
the level found in adult swallows because of its 
low calculated costs (Klasing 2004). Some 
components of innate immunity (e.g.. natural 
antibodies, complement mediated lysis and lym¬ 
phocytes) are lower in nestling Tree Swallows 
than in adults, but lymphocyte concentrations 
reach adult levels in 18-day old nestlings 
(Palacios et al. 2009). 
We investigated the relationship between 
physical growth and maturation of immunity from 
measurements of mass and right wing chord 
length when we took blood samples. We assessed 
the heritability of the immune response by 
comparing the in vitro microbicidal ability of 
whole blood to kill E. coli in mothers and their 
offspring. 
METHODS 
Study Species .—We studied Tree Swallows that 
nested on the campus of Grand Valley State 
University in Allendale, Michigan. USA (42 57' 
N, 85 53' W) from May to July 2009. Our study 
site in a fallow agricultural field consisted of a 10 
X 10 grid of 100 standard wood nest-boxes 
spaced 20 m apart and fitted with predator guards. 
Tree Swallows are primarily aerial insectivores 
with a socially monogamous breeding system 
characterized by high rates of extra-pair paternity 
(Robertson el al. 1992). Females usually lay 
clutches of 5-6 eggs that hatch after -14 days 
of incubation. Hatchlings are altricial and both 
adults tending the nest provision nestlings until 
most fledge between 18 and 20 days after 
hatching (Robertson et al. 1992). 
Field Methods.—We monitored nests every day 
to ascertain clutch completion dates, exact 
hatching dates, and exact nestling ages. Tree 
Swallow nestlings undergo three basic stages of 
development: their eyes open 3^4 days after 
hatching, they develop endothermy 8-9 days after 
hatching (Dunn 1979), and they fledge ~2() days 
after hatching (Robertson et al. 1992). We 
collected blood from different nestlings on 
nestling days (ND) 6, 12. and 18 (ND0 - the 
day the first egg in a clutch hatched) that are in the 
range of these developmental stages; nestlings 
were too small at ND3-4 to obtain blood samples 
o adequate volume to perform in vitro assays. 
ohlv’ r !° tbe smal1 sizc of nestlings, we 
obtained only a single blood sample from each 
individual to prevent jeopardizing its health. 
NDI8 was the end-point measurement because 
some nestlings may fledge before ND20 and we 
wanted to ensure collecting blood samples while 
nestlings were available. We collected Wood 
samples from 10 nestlings from five broods on 
ND6. 16 nestlings from five broods on ND12.and 
22 nestlings from eight broods on ND18. We did 
not measure the ND6 nestlings from which we 
drew blood, but we did measure mass to the 
nearest 0.1 g on an electronic scale and flattened 
right wing chord to the nearest I mm with a ruler 
with a stop fixed to one end on 118 ND6 nestlings 
at 25 other nests at our study site. We measured 
nestling mass on ND12 and ND18 to the nearest 
0.2 g with a spring scale and flattened right wing 
chord ol the nestlings from which we drew blood 
samples. 
Adult swallows were captured during Ihe 
nestling phase of the breeding season using plastic 
box-traps (Yunick 1990). We drew blood front 79 
adults (n = 44 females, n = 35 males). We 
measured each adult’s mass and right wing chord 
length. Breeding females were categorized as 
either second-year (SY) (n = 19) if the female had 
a mostly brown dorsal plumage, or after-second 
year (ASY) (n - 25) if the female had mostly 
iridescent blue-green dorsal plumage (Hussell 
1983). Male Tree Swallows cannot be reliably 
classified to age by size or plumage characteristics 
(Dwight 1900. Robertson et al. 1992). but males 
mated to ASY females are likely to be ASY males 
(Robertson et al. 1992). 
We drew blood (10-60 pL) from the brachial 
vein ol swallows. The area surrounding the 
brachial vein was cleared of interfering feathers, 
soaked liberally with 70% ethanof (EtOH). 
sw'abbed with a fresh cotton ball, and allowed to 
air dry for 15-20 sec because EtOH can cause 
hemolysis, which can complicate immune assays 
(Millet et al. 2007). The brachial veins of 
nestlings and adults were punctured with either 
sterile 28-gauge hypodermic needles or lancets, 
respectively, and blood was collected in heparin¬ 
ized capillary tubes (50 pL capacity). Tubes were 
held horizontally to cause air bubble formation al 
the end of the tube, sealed using an EtOH- 
sterilizcd clay card, placed in sterile 50 mL test 
tubes, and transported to the laboratory. All blood 
samples u r ere collected within 3 min of handling 
to avoid the effects of immunosuppression 
mediated by stress hormones such as corticoste¬ 
rone (Romero and Romero 2002). Blood was 
