Humoral immunocompetence in relation to condition, size, asymmetry and MHC class II variation in great snipe (Gallinago media) males
Robert Ekblom, Dennis Hasselquist, Stein Are S{\Ae}Ther, Peder Fiske, John Atle K{\Aa}L{\Aa}S, Mats Grahn, Jacob HÖglund
1 Humoral immunocompetence in relation to condition, size, asymmetry and MHC class II variation in great snipe (
Gallinago media ) males
R. EKBLOM *, D. HASSELQUIST , S. A. SÆTHER
1, 3 , P. FISKE , J. A. KÅLÅS , M. GRAHN and J. HÖGLUND Population Biology/Evolutionary Biology Centre, Uppsala Uni-versity, Norbyv.18D, SE-752 36 Uppsala, Sweden 2.
Department of Animal Ecology, Lund University, Ecology Build-ing, SE-223 62 Lund, Sweden 3.
Evolutionary Biology/Evolutionary Biology Centre, Uppsala Uni-versity, Norbyv.18D, SE-752 36 Uppsala, Sweden 4.
Norwegian Institute for Nature Research, Tungasletta 2, N-7485, Trondheim, Norway 5.
Södertörn University College, Box 4101, SE-141 04 Huddinge, Sweden *e-mail: [email protected]
Short title: Immunocompetence in great snipe Summary 1.
In recent years many studies have investigated the relationships between different aspects of the immune system and ecology in various organisms. Yet, it remains unclear why individuals differ in their ability to mount an immune response against various antigens (often referred to as “immuno-competence”). Different kinds of trade-offs may be involved and costs of mounting the immune response often lead to condition dependent effects. We investigated how variation in condition, morphology and genetic vari-ables influenced the amount of antibodies produced against two novel anti-gens in a migrating bird, the great snipe (
Gallinago media ). We found no evidence for condition dependence of the antibody response and no effect of MHC genetics. There was, however, a weak negative corre-lation between body size and the amount of antibody production, which may indicate a trade-off between growth and immune response in this species.
Key words : Humoral immunocompetence, MHC Class II B, Bird, ELISA, DGGE 3
Introduction
During the last decade much research has been conducted in the field of “immunoecology” (Sheldon & Verhulst 1996; Westneat & Birkhead 1998; Owens & Wilson 1999). The immune system of both vertebrates (Norris & Evans 2000) and invertebrates (Ryder & Siva-Jothy 2000; Cotter, Kruuk & Wilson 2004) has been used to address important ecological and evolution-ary issues such as life history decisions (Norris & Evans 2000; Cotter et al. et al. et al. et al. et al. et al.
Somateria mollisima ; Hanssen et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.
Gallinago media ). We used two different harm-less antigens (diphtheria and tetanus combined in a vaccine for normal use in humans) and followed specific antibody production to each of these anti-gens. Furthermore we studied the MHC genetics of great snipe by investigat-ing the entire second exon of the MHC class II B gene (Ekblom, Grahn & Höglund 2003). 5
Materials and methods
FIELD STUDY The great snipe is a migrating wader species, breeding in northern Europe and wintering in Africa. Our study site is located in the Dovre mountains in central Norway (for a general description of the species and the field site see Løfaldli, Kålås & Fiske 1992; Fiske & Kålås 1995). Male birds display in groups on leks from mid May to the beginning of July. We captured birds with mist-nets on the leks in the end of May and beginning of June in the years 2001 and 2002. Each captured male was ringed and we also collected blood and morphological data before releasing it. See Höglund et al. (1990) for information on methods used for measuring morphometry. Data from 51 males is included in this study.
MEASUREMENT OF THE ANTIBODY RESPONSE We measured the amount of antibodies to two novel antigens (diphtheria and tetanus toxoid) by injecting males on the leks with 100 µl of diphtheria and tetanus vaccine. Before the vaccination, a control blood sample was taken from each bird. After approximately 12 days the birds were re-caught and a response blood sample was taken (the number of days varied from 11 to 15 but 45 out of 51 males were re-caught after exactly 12 days). Plasma was extracted from the blood after centrifugation and the amount of antigen-specific antibodies in the plasma was measured using an enzyme-linked im-munosorbent assay (ELISA) procedure. For a more detailed description of the immunisation and immunoassay see Hasselquist et al. (2001, 1999) and Ekblom et al. (in press B). All antibody concentrations was measured as the slope of the substrate conversion over time measured in the units 10 -3 × opti-cal density per minute (mOD/min) (measured on a V max ELISA plate reader, Molecular Devises, Sunnyvale CA, and analyzed using KineticCalc soft-ware, Winooski), with a higher slope indicating a higher concentration of specific antibodies in the sample. Control and response samples were run in duplicate on the ELISA reader and the average of these were used as anti-body titers in all analyses. Antibody response was defined as antibody titer in the response sample minus the antibody titer in the control sample. The levels of antibodies in the control samples differed between the two years and the responses in 2002 were corrected for these differences (see Ekblom et al in press B). et al. et al. in press A, for a description of the molecular methods). Sequences were analysed using BioEdit 5·0·9 (Hall 1999) and MEGA 2·1 (Kumar et al. et al. in press A).
Table 1.
Number of nucleotide differences within and between the six different allelic lineages of MHC class II B sequences used in this study. Standard Error (within parentheses) was calculated from 1000 bootstrap replications.
Name of allelic lineage Number of alleles included Mean number of nucleiotide differences within lineage Mean number of nucleotide differences between lineages a
2 1·00 (0·93) 13·13 (3·16) b
3 5·33 (1·72) 11·83 (2·75) c
4 3·67 (1·32) 10·03 (2·53) d
5 2·80 (1·20) 9·04 (2·36) e
2 6·00 (2·33) 10·14 (2·51) f
5 7·00 (1·81) 12·75 (2·81)
STATISTICAL ANALYSES Because the distributions of the antigen responses were highly skewed the values were log transformed before analyses. All probability values are two-tailed. For statistical analyses we used SPSS 11·5. 7 VARIABLES IN THE REGRESSION ANALYSIS
Antibody response : We combined the variables for the antibody response to diphtheria and tetanus vaccine using a principal component analysis (PCA) (see Kilpimaa, Alatalo & Siitari 2004). The first principal component ex-plained 70·4% of the variance and was used as a measure of total antibody response in further analyses. The values of this variable did not differ from a normal distribution (Kolmogorov-Smirnov test, Z = 0·726, n = 51, p = 0·67).
Size : We calculated body size as the first principal component of 3 different morphological measurements (tarsus length, total head length and wing length). This variable explained 61·6% of the variance of the three measure-ments.
Condition : Condition was calculated as the residuals of a regression between body mass and size (Brown 1996) controlled for time of the night and date, both of which are known to be correlated with body mass (Höglund, Kålås & Löfaldi 1990). This regression was highly significant (F = 16·97, p < 0·001).
Asymmetry : As a measurement of body asymmetry we used the first princi-pal component of the absolute difference of left side and right side values of three different two sided measurements (tarsus length, wing length, and the amount of white on tail of the outermost tail feather; Fiske, Kålås & Sæther 1994). This variable explained 48·8% of the total absolute differences be-tween these traits.
Whiteness on the tail : As a measure of the amount of white on the tail (a trait possibly subjected to sexual selection; Höglund, Eriksson & Lindell 1990; Sæther et al. et al. et al.
MHC heterozygosity : Heterozygosity in the MHC class II B gene was ap-proximated as the number of alleles (one to three) found in each individual using the PCR based typing system (see Genetic Analyses).
Age : In this species it is possible to determine whether a caught bird is one year old or older by determining the feather wear (Sæther, Kålås & Fiske 1994). We used minimum estimate of age, where an old bird caught for the first time was assumed to be two years old. 8
Days to resampling : The number of days between immunisation and re-sampling was also included in the analysis.
Results
RESPONSE TO IMMUNISATION In 2001, we immunized 59 males and 33 of these were re-caught. In 2002 the corresponding figures were 24 re-caught out of 47 vaccinated. Birds re-sponded to the vaccination by producing specific antibodies to each of the two antigens (Paired t-test, tetanus, t = -11·943, df = 50, p < 0·001; diphthe-ria, t = -4·625, df = 50, p < 0·001). Six individuals were immunized and re-caught in both years of the study, these did not produce a higher antibody titer after the second vaccination as compared to their primary immune re-sponse (Paired t-test, tetanus, t = -0·142, df = 5, p = 0·893; diphtheria, t = -1·712, df = 5, p = 0·148; Unpaired t-test, tetanus, t = 0·938, df = 55, p = 0·352; diphtheria, t = -0·027, df = 55, p = 0·978, Fig. 1). The responses of these six individuals tended to be positively correlated for the two study years, even though this was not significant, probably due to the low sample size (tetanus, r = 0·792, n = 6, p = 0·060; diphtheria, r = 0·601, n = 6, p = 0·207). Only the primary immune response from each individual was used in further analyses.
Fig. 1.
Strength of the immune response (± SE), as measured by the difference in antibody titer between control sample and response sample. The response to tetanus is higher than the response to diphtheria. The response to a secondary injection was not stronger than to the primary injection. = 0·408, n = 51, p = 0·003, Fig. 2) and the response was stronger to tetanus than to diphtheria (Paired t-test, t = -7·233, df = 50, p < 0·001, Fig. 2). This relationship appeared to be non-liear and individuals that responded strongly to diphtheria almost always had a strong tetanus response whereas individuals with a strong tetanus response could have any kind of response against diphteria (see Fig. 2). In further analyses, we used a combination of the two responses (from a PCA, see methods). There were no differences in antibody response between the two years of this study (t = -1·041, df = 49, p = 0·303). -2-1.5-1-0.500.511.52
Log (diphtheria+1)
Log t e t a nu s Fig. 2.
Relationship between antibody responses to diphtheria and tetanus antigens (for statistics see text).
IMMUNE RESPONSE AND MHC TYPE We did not find any differences between the allelic lineages in the amount of antibody production (ANOVA, F = 1·10, p = 0·36). We also made an-other type of analysis, grouping antibody response into either high response, defined as one or both of the two responses being higher than 1·5 mOD/min, or low response, if both were lower than 1·5 mOD/min. Still there was no difference in how many times alleles of the different lineages were present in individuals from the two groups (Fisher’s exact test, p = 0·64). We also ana-10 lysed whether there was any effect on the antibody response that could be caused by the combination of allelic lineages (genotype) in an individual. Again, no such effect was found (ANOVA, F = 0·90 p = 0·59) and there was no effect on the antibody response of the number of MHC alleles (het-erozygosity) (ANOVA, F = 0·94 p = 0·40).
Table 2.
Linear regression of the humoral immune response (antibody titer) in the great snipe.
Variable B Std Error Standardised Beta t p (Intercept) 2·82 3·07 0·92 0·36 Size -0·37 0·16 -0·37 -2·34 0·024 Condition 0·005 0·030 0·027 0·16 0·87 Asymmetry 0·032 0·15 0·032 0·21 0·84 White tail -0·007 0·054 -0·021 -0·13 0·90 MHC heterozygosity 0·18 0·28 0·098 0·66 0·52 Age 0·068 0·13 0·081 0·50 0·62 Days to re-sampling -0·27 0·24 -0·17 -1·13 0·27
CONDITION DEPENDENCE OF THE IMMUNE RESPONSE We analysed possible effects of condition-dependent traits and MHC het-erozygosity on the antibody response, using a linear regression. The only variable with an significant effect was size (Table 2) and the full model was not significant (R = 0·14, F = 0·99, p = 0·45). A model with size as the only variable was significant (R = 0·10, F = 5·44, p = 0·024, Fig. 3). There was a weak negative correlation between size and immune response (r = -0·32, n = 51, p = 0·024, Fig. 3). This correlation was significant for teta-nus (r = -0.32, n=51, p=0.022) but not for diphtheria (r = -0·21, n = 51, p = 0·14). Discussion
In this study of the humoral immune response in the great snipe, birds re-sponded to vaccination with diphtheria and tetanus toxoid by producing spe-cific antibodies to both antigens used. There was a correlation between the responses to the two antigens and in particular, individuals responding strongly to diphtheria also responded strongly to tetanus. This pattern is very similar to that of other studies that have used these two antigens (Svensson et al. et al.
Size 3210-1-2-3 I mm une r e s pon s e Fig. 3.
Relationship between size and immune response in great snipe males. There is a weak negative correlation (see text) between these traits suggesting a possible trade-off. The regression line (Y = -0·32X) from the linear regression is included.
A few of the birds were caught and vaccinated in both years of study. The correlation between the antibody response for the two years of these indi-viduals (although not statistically significant due to small sample size) sug-gest that the strength of the antibody response is stable within individual birds even after one year. We found no indication that repeated vaccination caused a stronger sec-ondary response. In vertebrates, the general pattern is that secondary anti-body responses are much higher than primary responses (e.g., Roitt 1997), and this has also been found in many other studies of wild birds repeatedly exposed to an antigen (Nordling et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.
Acknowledgements
We thank S. L. Svartaas for field assistance and D. Sejberg, G. Engström and B Rogell for lab assistance. Permissions for capture, ringing, blood sam-pling and immunisation of birds were given by Stavanger Museum and the Norwegian Animal Research Authority. Financial support were given by Helge Ax:son Johnsons
Stiftelse and Stiftelsen för zoologisk forskning (to R.E.), the Swedish Research Council for Environment, Agricultural Science and Spatial Planning (Formas), Carl Tryggers Stiftelse, Crafoordska Stiftel-sen and Magn. Bergvalls Stiftelse (to DH), the Research Council of Norway (to SAS) and the Swedich Research Council, VR (to JH).
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