DOI: 10.5061/dryad.4xgxd25br
title: Data and code for: No evidence of metabolic costs following adaptive immune activation or reactivation in house sparrows

description: We examined the energetic costs of immune activation by measuring both
 basal (BMR) and exercise-induced maximal metabolic rates (MMR) in house
 sparrows before and after being injected with either saline (sham) or two
 novel antigens (keyhole limpet haemocyanin and sheep red blood cells; KLH
 and SRBC, respectively) after the primary and two subsequent vaccinations.
 We also examined the effect of experimentally-induced breeding levels of
 testosterone (T) on immune responses and their metabolic costs
 in both males and females. 
description: <em>Experimental animals</em> We
 captured free-living house sparrows (<em>Passer
 domesticus</em>) residing in the Illawarra region of NSW. The 31
 males and 31 females were distributed among 4 outdoor flight cages (4.5 x
 3.6 x 2.5 m) exposed to ambient conditions. Birds had free access to
 commercial finch seed mix (Golden Cob, Mars Birdcare Australia),
 mineralised grit, and water. <em>Experimental
 protocols</em> All male
 birds were bilaterally castrated, under anaesthetic, one or two days after
 initial capture and returned to the flight aviaries.  Birds were allowed
 two weeks to adjust to captivity before measuring their basal and
 exercise-induced maximal metabolic rates (BMR and MMR, respectively).
 Blood was then sampled to characterise hormonal and immune status, and
 each bird implanted with either an empty or testosterone-filled silastic
 tubule (see below). Metabolic evaluations and blood sampling were
 undertaken 2 weeks later to determine T levels and immune response
 parameters (below). Based on these MMR rankings, male and female birds
 were distributed randomly into 4 experimental groups: antigenic
 injection/testosterone implant (IC(immune challenge)/T), antigenic
 injection/control implant (IC/C), sham injection/testosterone implant
 (S/T), sham injection/control implant (S/C).
 <em>Testosterone implant procedure</em>
 Birds were implanted subcutaneously with either an empty silastic
 capsule (control birds) or a silastic tube filled with crystalline
 testosterone (Sigma Chemical Co.; T-1500). The capsules were prepared from
 6 mm lengths of medical-grade silastic tubing (1.47 mm inner diameter;
 1.96 mm outer diameter; Dow Corning), which were sealed at each end with
 silastic glue (Dow Corning). Birds were given anaesthetic (methoxyflurane)
 and had a small incision made to permit subcutaneous insertion of implants
 on one side of their thorax. The resultant wounds were sealed with
 medical-grade cyanoacrylate adhesive (Vetbond).
 <em>Immune challenge through
 hypervaccination</em> Two novel antigens, keyhole
 limpet haemocyanin (KLH; Sigma-Aldrich) and sheep red blood cells (SRBC;
 Institute of Medical and Veterinary Science, Adelaide, South Australia)
 were used to elicit adaptive immune responses in the immune-challenge
 groups. Inoclation using KLH followed Hasselquist et al. (1999
 <em>Behavioral Ecology and Sociobiology</em>), with 1 mg
 KLH/ml sterile water emulsified 1:1 with Freund’s incomplete adjuvant
 (Sigma-Aldrich) for the immune challenge groups (IC/T and IC/C) and
 sterile water emulsified 1:1 with Freund’s incomplete adjuvant for the
 sham challenge groups (S/C and S/C).  Birds were injected with 100 ml of
 either mixture into their pectoral muscle. Immune-challenged birds were
 also injected intra-abdominally with 100 ml of SRBC (10% by volume in
 phosphate-buffered saline (PBS)), whereas sham-challenged birds were
 injected with 100 ml PBS. These injections were administered three times,
 2-3 weeks apart, to achieve hypervaccination. Metabolic measurements and
 blood samples were collected 12 days after the first injection and 6 days
 after the second and third injections.
 <em>Quantifying adaptive immunity</em>
 Following venepuncture, ~200 μl of blood was collected from an
 alar vein in heparinised microhaematocrit tubes. Blood samples were
 immediately centrifuged at 6000 rpm for 5 min and plasma stored at -20 ˚C
 for hormonal (see Supplementary material) and immune analyses. Plasma
 concentrations of anti-KLH antibodies were determined using enzyme-linked
 immunosorbent assay (ELISA) described by Hasselquist et al. (1999
 <em>Behavioral Ecology and Sociobiology</em>) and Martinez et
 al. (2003 <em>Functional Ecology</em>). Constitutive innate
 immunity and specific immunity to SRBC were determined by quantifying
 agglutination and lysis activity of plasma before and after exposure to
 SRBC, respectively, following Matson et al. (2005 <em>Developmental
 and Comparative Immunology</em>), modified to use 1% suspension of
 SRBC instead of rabbit RBC. <em>Basal metabolic
 rate </em> Measurement of BMR was conducted when
 birds were post-absorptive, during the birds’ rest-phase, and at
 temperatures within the thermoneutral zone for this species (29-31˚C). At
 least 3 h after they had last fed, birds were placed individually in
 2-litre metal chambers fitted with a perch and supplied with room air at
 500 ml min<sup>-1</sup> (Tylan mass flow controllers FC-280S).
 Oxygen content of inlet and outlet air for each chamber was measured using
 a Sable Systems Oxzilla II oxygen analyser in combination with an
 electronic stream selector (Sable Systems Respirometer Multiplexer V 2.0).
 Basal rates of oxygen consumption (BMR) were defined as the mean of the
 two lowest 5-minute periods of oxygen uptake recorded over the 12-hour
 measurement period. <em>Maximum metabolic rate
 </em> Oxygen consumption rates ( O<sub>2</sub>) were measured during intense exercise within an enclosed 5-litre drum with clear sides and carpet lining the inner rim (18). A mass-flow controller (Tylan Corp.) supplied air to the chamber at 5 l min<sup>-1</sup> and the oxygen content of inlet and outlet ports was measured with an oxygen analyser (Sable Systems FC-1).  A single bird was placed in the drum and, once settled, the cover was removed and the drum rotated. The Ping-pong balls within the drum encouraged birds to maintain a series of rapid takeoffs and short-term flights interspersed with vigorous hopping. The  O<sub>2</sub> data were adjusted with ‘instantaneous’ conversion procedures to account for gas mixing characteristics of the wheel and accurately resolve short-term oxygen content variation. The highest continuous 60-sec instantaneous oxygen consumption rate was designated as MMR. <em>Statistical analysis</em> Immune parameters after each of the three injection stages were quantified as individual’s change, positive or negative, in a given parameter compared to their ‘baseline’ for that parameter. Changes were expressed as a difference above or below an individual’s baseline value (e.g. KLH<sub>change@stage1</sub> = KLH<sub>baseline</sub> – KLH<sub>stage1</sub>). Metabolic parameters were likewise expressed as a departure from baseline (e.g. BMR<sub>proportion@stage1</sub> = BMR<sub>stage1 - </sub>BMR<sub>baseline</sub>).
description: Description of the data variables and any missing values can be
 found in the README file.  Please read the README file
 and contact the authors if you would like to reuse this
 dataset.