Behavioral Ecology Advance Access originally published online on August 11, 2004
Behavioral Ecology 2005 16(2):497-498; doi:10.1093/beheco/arh137
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The spatial and temporal repeatability of PHA-responses
Department of Animal Ecology, Lund University, S-223 62 Lund, Sweden
Address correspondence to M. Granbom. E-mail: martin.granbom{at}zooekol.lu.se.
Received 7 January 2004; revised 25 May 2004; accepted 1 June 2004.
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INTRODUCTION |
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 TOP INTRODUCTION MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The capacity to mount a cell-mediated immune response in birds is often quantified as the swelling following an injection of phytohaemagglutinin (PHA) into the wing-web. Most studies have found the repeatability of consecutive measurements of the same swelling to be high (e.g., Alonso-Alvarez and Tella, 2001
; Fargallo et al., 2002; Saino et al., 1997; Smits et al., 1999; Tella et al., 2000). However, as pointed out by Siva-Jothy and Ryder (2001), repeatability calculated this way only estimates the precision with which an experimenter measures the size of a particular swelling; it does not encompass other components of measurement error. Furthermore, such estimates of repeatability do not capture temporal variation in individuals' PHA-responses. To our knowledge, no study has reported the latter types of repeatability estimates. Thus, the aim of the present study was to investigate the repeatability of the response to PHA both at a given time and between different occasions separated in time. |
MATERIALS AND METHODS |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We investigated the immune responsiveness of European starling (Sturnus vulgaris) nestlings and fledglings in the Revinge area east of Lund, Sweden (55° 42' N, 13° 28' E). Birds were injected with a 0.1 ml PHA solution (1 mg in 100 µl PBS) subcutaneously in the middle of the wing-web. Prior to injection, the injection site was marked with a waterproof pen and measured three times to the nearest 0.001 mm. The final three measurements were taken 24 h ± 10 min later. The same person (L.R.) handled all birds and another person (M.G.) did all injections and measurements. Measurements were taken with a digital micrometer (Mitutoyo, no. 293–801), which was tightened until the skin started to twist. Between each measurement taken, the micrometer was removed completely from the wing. By matching readings 1, 2, and 3 pre-injection with readings 1, 2, and 3 after injection, respectively, three different repeated measures of the same swelling were obtained.
To estimate ‘spatial’ repeatability, 18 nestlings from four nests were injected with PHA in both wing webs on day 11 after hatching. At this age, starling nestlings have reached asymptotic mass (mean body mass = 74.0 g) and tarsus length, whereas their feathers are still growing (Feare, 1984; Smith and Wettermark, 1995).
To estimate ‘temporal’ repeatability, we measured immune responsiveness of starling fledglings housed in outdoor aviaries in the study area. Twenty-day old nestlings were taken into aviaries and kept there throughout this study. Thirty birds were injected in both wing-webs with PHA at the age of 50 days (± 3 days) and again 46 days later. Because of an unsuccessful injection, measures of one swelling were excluded from subsequent analyses.
Repeatability is the proportion of variance in a measured quantity that occurs among, rather than within individuals (e.g., Becker, 1984; Lessells and Boag, 1987). We estimated repeatability, as well as several different components of measurement error, using nested ANOVAs where the among-individuals variance components represent repeatabilities. The variance components were estimated using restricted maximum likelihood (Proc Mixed, SAS 8.02, Littell et al., 1996). For unbalanced data, F-tests were performed using Satterthwaite's approximation.
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RESULTS |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spatial repeatability
The mean swellings were 1.12 mm ± 0.30 (mean ± SD) and 1.16 mm ± 0.29 in the right and left wings, respectively. Most of this variation was caused by variation among individuals, but close to a third of the variation was caused by variation among wings within individuals (Table 1). The proportion of measurement error caused by differences between wings was more than twice that caused by differences among micrometer readings within wings (Table 1). The overall repeatability of PHA swelling was 0.58.
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Temporal repeatability
The mean swelling at the age of 50 days was 0.83 ± 0.22 and 0.98 ± 0.31 mm in the right and left wings, respectively. The corresponding measures at the age of 96 days were 1.14 ± 0.44 and 0.97 ± 0.25 mm. In nested ANOVAs, results will depend on the order in which terms are nested. Because the similarity between subsequent swellings on the same wing (mean r = 0.14) was not greater than that for swellings on alternative wings (mean r = 0.19), we chose to nest wing under time (Table 2). The variance among wings within time periods and individuals accounted for more than half of the total variance, and variance between time periods accounted for an additional fifth of the total variance (Table 2). The estimated repeatability of PHA swelling was as low as 0.14.
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DISCUSSION |
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TOPINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We measured the size of a particular swelling following an injection with PHA with the same high precision as in previous studies (e.g., Alonso-Alvarez and Tella, 2001
; Brinkhof et al., 1999; Fargallo et al., 2002; Forero et al., 2001; Saino et al., 1997; Soler et al., 1999; Tella et al., 2000, 2001; Velando et al., 2001). However, the accuracy with which a particular swelling is measured cannot be equated with the repeatability of the immunological reaction. Since the aim of measuring swellings is to obtain an estimate of immunological capacity, variation between different swellings obtained at the same time on the same individual should also be considered when calculating repeatability. Repeatability calculated this way was low (0.58) compared with that of, for example, morphological traits of birds (Boag and van Noordwijk, 1987). The low repeatability may be explained by, for example, variation in the exact location of injection in the patagium. If our estimate is typical of studies of passerine birds, it limits the effect size that can be expected in such studies. We recommend that future studies using the PHA technique report an estimate of the spatial repeatability, in addition to the usual estimate of the accuracy by which a particular swelling is measured, and that this value is taken into account when interpreting the results (in particular when they are negative).
The PHA-response also varied temporally, resulting in a low repeatability over time. This may be caused by short-term variation in phenotypic condition (Lifjeld et al., 2002; Zera and Harshman, 2001). A study of Zebra finches has shown that the activity immediately prior to injection may even affect the strength of the response (Ewenson et al., 2003). A low temporal repeatability may help explain the low heritability of this trait found in several previous studies (Brinkhof et al., 1999; Christe et al., 2000).
In conclusion, the low spatial and temporal repeatability of the PHA-response increases the risk of type II errors in studies of immunoecology. To increase the precision of estimates of immune responsiveness, we suggest that both wings are injected with PHA and that subsequent calculations are based on the mean response in both wings. In our case, this would increase the repeatability from about 0.58 to around 0.73 (cf. Falconer and Mackay, 1996). It should, however, be noted that the starling is a relatively large passerine and that the precision may be lower when working with smaller passerines. Furthermore, by injecting PHA into both wings one necessarily sacrifices a control treatment. Although Smits et al. (1999) showed that there is no effect of injecting PBS alone, the lack of control could be a problem when PHA responses are measured during growth and if nestlings differ in growth rate. Finally, the low temporal repeatability should be kept in mind in studies of selection and heritability of responsiveness to PHA and when using PHA response to assess variation in individual quality.
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ACKNOWLEDGEMENTS |
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This study was partly financed by a grant from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning to H.G.S. We thank an anonymous reviewer and, in particular, Göran Arnqvist for comments that greatly improved the manuscript. We also thank Johan Blom for interesting discussions on immunology.
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