Sexual selection has attracted considerable empirical and

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1 Behavioral Ecology Vol. 7 No. 2: Sexual ornamentation and immunocompetence in the barn swallow Nicola Saino" and Anders Pape M0ller 1> a Dipartimento di Biologia, Sezione di Zoologia, Scienze Naturali, Universita di Milano, Via Celoria 26, Milano, Italy, and b Department of Population Biology, Copenhagen University, Universitetsparken 15, DK-2100 Copenhagen 0, Denmark The handicap hypothesis of honest signaling suggests that secondary sexual characters reliably reflect phenotypic or genotypic quality of signalers. This hypothesis is based on the assumptions that signals are costly to produce and/or maintain and the cost of a given level of signaling is higher for low quality than for high quality signalers. We tested these assumptions in a field experiment in which the size of a secondary sexual character [tail length in male barn swallows (Hirundo rustica)] was experimentally manipulated. Males were randomly assigned to tail elongation, tail shortening, or two control treatments (tail manipulation, or just capture, ringing, and handling). Male barn swallows were challenged with an injection of sheep red blood cells, and blood was sampled on the day of first capture and after 3 to 4 weeks for determination of concentrations of gammaglobulins. Tail-elongated males did not increase levels of gamma-globulins while males of the other three groups demonstrated increases. Analyses of variation in gamma-globulins within treatment groups revealed a positive correlation between gammaglobulins and original tail length among males with elongated tails. These results suggest that tail length imposes an immunocompetence cost on males, and that males with naturally long tails are differentially better able to cope with this cost. Key words: barn swallow, gamma-globulins, Hirundo rustica, immunocompetence, sexual selection, tail ornament. [Behav Ecol 7: (1996)] Sexual selection has attracted considerable empirical and theoretical interest from evolutionary biologists during the last decades, and current reviews suggest that strong mate preferences for extravagant secondary sexual characters are widespread throughout the animal kingdom (reviews in Andersson, 1994; M0ller, 1994). There is much less concensus concerning the nature of fitness benefits accruing to choosy individuals, although it is generally agreed that females may obtain both direct and indirect fitness benefits (reviews in Andersson, 1994; Moller, 1994). Reliable signaling or handicap theory suggests that secondary sexual characters reliably reflect phenotypic (or genotypic) quality of signalers because of differential costs of signals (Grafen, 1990a,b; Heywood, 1989; Iwasa et al., 1991; Nur and Hasson, 1984; Zahavi, 1977). Only males in prime condition are able to cope with the costs of an extreme signal, and for a given level of display, low-quality individuals experience a higher cost than high-quality ones. Reliability is hence enforced by this differential cost with respect to phenotype. Receivers of signals may thus obtain reliable information on the ability of signalers to cope with the various costs imposed by the production or maintenance of the signal. Individuals developing a secondary sex trait thus have to optimize their level of signaling. Only males in prime condition will be able to produce an extravagant sex trait without compromising their ability to cope with several potential costs imposed by the trait. Here we present the first experimental test of the ability of males to respond to a challenge to their immune system in relation to exaggeration of ornamental secondary sexuaj characters in a bird with an intense, directional mate preference. The barn swallow (Hirundo rustica) is a small, socially monogamous, insectivorous passerine with a current directional female mate preference for males with long outermost tail feathers (review in Meller, 1994). Males Received 6 March 1995; revised 24 July 1995; accepted 2 August /96/$ International Society for Behavioral Ecology with long tails have lower prevalences and intensities of several ectoparasites (Moller, 1991; Saino and M0ller, 1994), and resistance to infections with hematophagous mites appears to have an additive genetic component as shown by partial crossfostering experiments (M0ller, 1990). Levels of immune system activation, as reflected by the size of immune organs, is inversely related to the degree of tail exaggeration in male barn swallows (Moller et al., 1996). Furthermore, long-tailed males appear to be better able to cope with a given increase in testosterone plasma level than short-tailed males as reflected in lower changes in leukocyte counts from manipulation to recapture (Saino et al., 1995). These results suggest that a long tail in male barn swallows reflects an efficient immune response, but does not specifically address the question whether long-tailed males have a better ability to raise an immune response toward a novel challenge to the immune system (Moller et al., 1996). The specific objectives of the present study were to experimentally test (1) whether a long tail imposes a cost on males in terms of ability to raise an immune response against a novel challenge; and (2) whether this immunocompetence cost varies differentially with respect to male phenotypic quality as reflected by the size of the secondary sexual character. These aims were addressed by experimentally manipulating the length of the outermost tail feathers and measuring the primary immune response to a novel challenge to the immune system of the males, produced by intraperitoneal injection of sheep red blood cells (Lochmiller et al., 1993; Tsiagbe et al., 1987). The differential immunodepressive cost was determined by relating the change in gamma-globulin levels, leukocytes counts, blood cell sedimentation rate, and hematocrit produced in response to the challenge to male phenotypic quality as reflected by their original tail length. Gamma-globulins are a heterogeneous class of plasma proteins that are the source of antibody proteins involved in humoral response to a large spectrum of parasite infestations, protozoa, bacteria, and viral infections and experimental pathogenic challenge (e.g., DeVaney and Augustine, 1988;

2 228 Behavioral Ecology Vol. 7 No. 2 Lochmiller et al., 1993; Soulsby, 1987). Total leukocyte count and concentration of different leukocyte families are one of the most important immunologicaj tests. Sedimentation rate depends on cell factors as well as plasma factors. It increases in a wide range of infectious and inflammatory diseases due to an increase of fibrinogen and gamma-globulins. Acute or chronic anemia as measured by hematocrit levels can result from blood and gastrointestinal parasite infestations or bacterial infections (Harrison and Harrison, 1986). Hence, the hematological variables we measured are likely to be good descriptors of both general health status and of humoral and cell-mediated immunocompetence of barn swallows, that is, their ability to mount an immune response to the experimental challenge to their immune system. STUDY AREA AND METHODS The study was carried out during spring 1994 on six farms located east of Milano (northern Italy). Male barn swallows started arriving at our study farms on March 27. Swallows were caught by mist nets at the time of their arrival at the colonies. Each barn swallow was marked with a metal ring on one leg and a plastic color ring on the other. Individuals were sexed according to the shape of the cloacal protuberance (Svensson, 1984), and this was later confirmed by inspection for presence (female) or absence (male) of an incubation patch and by observation of sexual and breeding behavior. At the time of first capture we measured several morphological variables including length of the left and right outermost tail feathers. Tail length was expressed as the mean length of the two outermost tail feathers. Individuals were weighed with a precision of 0.1 g on a Pesola spring balance. Body condition was expressed as body mass/(keel length)'. Blood samples (on average 180 u.1 in heparinized hematocrit capillary tubes) and smears were also taken at first capture and at time of recapture. Every time an individual was captured we estimated the intensities of infestation of three ectoparasite species according to the procedures outlined in Meller (1994). We assigned males sequentially to one of four experimental groups; the first, second, third, and fourth males to be captured were assigned to the first, second, third, and fourth group, respectively. The following males were assigned sequentially to the four experimental groups according to their order of capture. All males in these groups were injected intraperitoneally with 100 u,l of a PBS solution containing 5 X 10 5 sheep red blood cells (SRBC)/n-l of solution. This was used as a standard method to elicit an immune response in experimental males (Lochmiller et al., 1993; Tsiagbe et al., 1987). Males of the first group (hereafter II males) were given the above treatment and released. Both outermost tail feathers of males of the second group ( males) were cut at approximately 1 cm from the base and then reglued. Males of the third group (S males) had their outermost tail feathers shortened by 2 cm, while males of the fourth group (E males) had their tail elongated by 2 cm. The detailed methods of tail length manipulation are fully described in Moller (1988, 1994). We did not inoculate males with SRBC in every capture session. We used males that were not injected (C males) to estimate the change of the hematological variables considered in the natural population of males. The unbiased assignment of males to this group with respect to the four groups of males that received SRBC inoculation was confirmed by comparing the mean values of several potentially confounding variables (see Results). Sedimentation rate and hematocrit measures To measure sedimentation rate, heparinized capillary tubes in which blood samples were collected were put in vertical position for 4 h in a refrigerated room (4 C). Sedimentation rate (proportion of blood sedimented per hour) was expressed as volume of the part of the capillary not occupied by blood cells X blood volume in the capillary" 1 X Blood samples were then centrifuged for 10 min at 4000 rpm and plasma was stored at 30 C for gamma-globulin analysis. Hematocrit was expressed as volume of the part of the capillary occupied by blood cells X blood volume in the capillary" 1. Changes in sedimentation rate and hematocrit were expressed as the difference between values observed at recapture and values observed at first capture. Gamma-globulin assay Gamma-globulin assays were made, on the average, 3 months after blood collection by densitometric analysis after electrophoretic separation of plasma proteins on agarose gels (Paragon SPE kit, Beckman Instruments, Inc.). Five milliliters of plasma were diluted 1:2.5 in Barbital buffer (ph 8.6). Five milliliters of the diluted sample were applied to agarose gels following the standard procedures of the Paragon SPE kit. In one lane of each gel we also applied opportunely diluted chicken IgG to obtain a standard reference for migration of barn swallow IgG along the electophoretic lanes. The electrophoreses were applied at constant voltage (100 volts) at 20 C for 25 min. After electrophoresis, gels were air dried and stained following kit instructions. Densitometric analysis was performed by a computer image analysis procedure run by the Gelanalyst program (Eidosoft). The relative titer of gamma-globulin was expressed as the ratio between the area of the densitometric profile corresponding to the gamma-globulin region and the total area of the densitometric profile. Change of relative gamma-globulin levels (hereafter, change of gamma-globulin levels) between first capture and recapture was expressed as the difference between relative levels of gamma-globulin recorded in the blood samples collected at the time of recapture and that recorded at the time of first capture. Blood samples collected at the time of first capture and at the time of recapture were always applied to the same gel. Hence, accidental differences in analytical procedures between gels could not affect our measures of change of gammaglobulin levels between first capture and recapture. Within blood sample repeatability of relative gamma-globulin levels was highly significant (analysis of variance; F = 24.7, df = 7, 8, p <.001; see also Saino et al., 1995). Leukocyte counts Leukocytes and red blood cells were counted by an experienced person after blood smears had been air dried and stained by the May-Grunwald-Giemsa staining method. Blood smears were scanned at 630X magnification following standard routines. In each microscopic field we counted red blood cells and leukocytes classified as lymphocytes, monocytes, eosinophils, heterophils, and basophils [see Hawkey and Dennet (1989) for a photographic guide to identification of avian blood cells]. In each smear we counted 100 to 108 leukocytes, depending on the number of leukocytes present in the last microscopic field bearing leukocytes, and die corresponding red blood cells. This allowed us to calculate the relative frequency of leukocytes of each family with respect to the total population of leukocytes (hereafter, relative counts) and the number of leukocytes of the different families per 10,000 red blood cells (hereafter, absolute counts). This meth-

3 Saino and Moller Tail length and immunocompetence 229 Table 1 Mean ± SE change in relative gamma-globulin levels in a group of unmanipulated males (C), in two control groups inoculated with SRBC (II and ), and in two groups of inoculated males that had their tail either shortened (S) or elongated (E). Male treatment C S E Change in globulin n gammalevels ± (24.3 ± ) 6.7 ± (20.0 ± ) 7.7 ± 2.67 (18.5 ± 1.70) ± 2.26 (19.0 ± 2.27) * ( ± ± 2.61) Numbers in parentheses indicate mean ± SE values measured at first capture. " Changes that significantly differed from the change observed in II males (Bonferroni t test, p <.05). od has been shown to give significantly repeatable relative and absolute leukocyte counts (Saino et al., 1995). Change in relative or absolute counts of leukocytes has been expressed as the difference between counts recorded in the blood smears collected at recapture and those collected at first capture. Statistical tests, where not otherwise stated, were two-tailed. Difference in the change of gamma-globulin levels between II and C males was analyzed by one-tailed tests because there appeared to be no reason to expect that the change of gamma-globulin levels, as we defined it, was smaller in males that had been inoculated with SRBCs (II) to elicit an immune response than in uninjected C males. RESULTS First, we analyzed the relationships between degree of expression of tail ornaments (tail length) and hematological variables (relative and absolute leukocyte counts, gamma-globulin levels, sedimentation rate, and hematocrit) or intensity of infestation by ectoparasites [two species of Mallophaga {Machaerilaemus malleus and Myrsidea rustica) and one species of Diptera {Ornithomia bilobala)] as observed before any treatment was made, that is, at the time of first capture of males. We found no statistically significant correlations between degree of ornamentation and hematological variables (p values associated with Pearson's correlation coefficients always >.05). Tail length was significantly and negatively correlated with intensity of infestation by M. malleus (r =.19, p = 0.001, n= 305). We analyzed change in hematological variables among five groups of males. Males of three groups were inoculated with SRBC and had their tail either elongated, cut and reglued, or shortened. A fourth group was simply injected with SRBC, while the fifth included uninjected males. This allowed us to check if SRBC inoculation elicited an immune response, and, simultaneously, to check the effect of tail manipulation on the immune response. Males of the five groups did not differ in mean values of any of the variables potentially confounding the analyses of immune response. These included first capture date, a number of variables measured at the time of first capture such as leukocyte concentration, relative and absolute counts of leukocyte families (after angular transformation), gamma-globulin levels, blood cell sedimentation rate and hematocrit, body mass and body condition, intensity of ectoparasite infestations, and tail length (analysis of variance, p >.15 in all tests; results not shown). In particular, time elapsed from collection of the first and the second blood samples did not differ among the five groups (F =.79, df = 4,6, ns). Mean (SE) time to recapture, in days, was 27.6 (2.34) for C males, 24.9 (4.33) for II males, 21.3 (4.10) for males, 20.9 (3.30) for S males, and 22.8 (4.03) for E males (see Table 1 for sample sizes). Change in gamma-globulin levels significantly differed among the five groups of males (one-way analysis of variance, F = 4.83, df = 4,6, p =.001). We analyzed the difference in change of gamma-globulin levels between II males and C,, S, or E males by one-way ANOVA and the results were corrected for multiple comparisons by Bonferroni t tests (Sokal and Rohlf, 1969). Change in gamma-globulin levels was significantly larger in II than in C males (p <.05, Bonferroni one-tailed t test). Moreover, change in gamma-globulin levels was significandy larger dian zero in II males (H o : mean change = 0, t = 2.57, p < 0.01, one-tailed test), while it did not differ from zero in C males (M o : mean change = 0, t =.20, ns; Table 1). Hence, SRBC injection was effective in eliciting an antibody response in inoculated males. Change in gamma-globulin levels did not differ between II and males (p >.05, Bonferroni t test; Table 1) or between II and S males males (p >.05, Bonferroni t test; Table 1). However, II males showed a significantly larger increase in gamma-globulin levels than E males (p <.01, Bonferroni t test; Table 1). Apparently inoculation of SRBC did not affect differentially the change of leukocyte profiles, sedimentation rate, and hematocrit in the five groups of males. We found no significant difference in change of total concentration of leukocytes and of relative or absolute leukocyte counts (analysis of variance; p values always >.05; Figures 1 and 2). Similarly, change in sedimentation rate and hematocrit did not vary significantly among the five groups of males (F= 1.92, df = 4,5, ns, for sediementation rate; F = 1.96, df = 4,5, ns, for hematocrit; Figure 3). Antibody response and ornament size The relationship between change of gamma-globulin levels and premanipulation tail length was analyzed separately for each group by correlation analysis. Change in gamma-globulin levels was significantly and positively correlated with premanipulation tail length of tail-elongated males (Table 2 and Figure 4). However, we found no significant correlation between change in gamma-globulin levels and premanipulation tail length widiin each of the other groups of SRBC inoculated males (Table 2). DISCUSSION Natural levels of immune system activation were unrelated to die level of sexual signaling in die natural population of male barn swallows we studied. Hence, tiiere was little support for the idea that males widi a high degree of expression of ornamental traits consistently had better immune defense. The

4 Behavioral Ecology Vol. 7 No o 10-- phi 1 U "I B. n i I2 c M Male a t treatment Figure 1 Mean (with SE bars) relative leukocyte counts at first capture (open bars) and at recapture (full bars) in a group of unmanipulated males (C), in two control groups inoculated with SRBC ( and ), and in two groups of inoculated males that had their tail shortened (S) or elongated (E). Counts are expressed as percentage counts of each leukocyte family in a sample of 100 to 108 leukocytes. None of the leukocyte families showed significant difference in the change of relative counts among the five groups (one-way ANOVA, /"values always associated to p values >.05). Counts at first capture did not differ among groups. Sample sizes are 43, 21, 18, 22, and 24 for C, II,, S, and E males, respectively. lack of a positive correlation between length of tail ornaments and several hematological variables, and the negative correlation between intensity of infestation by M. malleus and tail length observed in the natural population of barn swallows are in agreement with findings from several previous studies (e.g., Meller, 1994; Saino et al., 1995). These results are consistent with the handicap theory because they show that high quality males with large ornaments had lower levels of parasite infestations, but apparently did not pay higher costs in terms of immune system activation than low quality ones. Sexual signals manipulation and immunocompetence costs Secondary sexual characters may be cosdy to produce or maintain and such costs may enforce reliability on sexual signaling (Grafen, 1990a,b; Heywood, 1989; Iwasa et al., 1991; Male S tre a tm e n t E Figure 2 Mean (with SE bars) absolute leukocyte counts at first capture (open bars) and at recapture (full bars) in a group of unmanipulated males (C), in two control groups inoculated with SRBC (II and ), and in two groups of inoculated males that had their tail shortened (S) or elongated (E). Counts are expressed as number of leukocytes per 10,000 red blood cells. None of the leukocyte families showed significant difference in the change of absolute counts among the five groups (one-way ANOVA, lvalues always associated to p values >.05). Counts at first capture did not differ among groups. See legend of Figure 1 for sample sizes. Zahavi, 1977). Hypothesized production costs include resources used for construction of the male trait and elevated metabolism, and reduced efficiency of the immune system caused by the immunosuppressive effects of elevated androgen profiles that promote the expression of ornamental traits (Folstad and Karter, 1992; Grossman, 1983). Possible maintenance costs include increased costs of locomotion and reduction of energy resources available for the competing demands of other metabolic activities (e.g., antibody production), re- o (A m m +

5 Saino and Meller'Tail length and immunocompetence re n=42 n=19 n=18 n=20 n=21 Table 2 Simple correlation coefficients between change of gamma-globulin levels and premanipulation tail length in a group of unmanipulated males (C), in two control groups inoculated with SRBC (II and ), and in two groups of inoculated males that had their tail shortened (S) or elongated (E). Male treatment Correlation coefficient n 65 -r n=45 C n=21 n=18 n=22 S n=24 E Male treatment Hgure 3 Mean (with SE bars) blood sedimentation rate and hematocrit at first capture and at recapture in a group of unmanipulatcd males (C), in two control groups inoculated with SRBC (II and ), and in two groups of males that had their tail either shortened (S) or elongated (E). See Methods for details on measurement of sedimentation rate and hematocrit. Change of sedimentation rate and hematocrit did not differ among groups (one-way ANOVA, F values always associated to p values >.05). Values at first capture did not differ among groups. duced foraging efficiency, and mortality costs arising from parasitism and predation (Evans and Thomas 1992; Magnhagen, 1991). We experimentally estimated the cost of a secondary sexual character, tail length, in terms of immunocompetence. Barn swallow males with manipulated secondary sexual signals were challenged by injections with SRBC, and subsequent immune responses were measured in terms of relative concentrations of gamma-globulins. Male barn swallows with elongated tails had significantly lower levels of gamma-globulins than males in the three other experimental groups, and they did not express a level of gamma-globulins above the basal level contrary to males in the other three groups (Table 1). Furthermore, uninjected males had significantly lower concentrations of gamma-globulins than injected males from the two control groups, indicating that there was an effect of injection with SRBC on immune defense. We checked for unbiased assignment to the four experimental groups of males and the group of uninjected males by comparing their initial measures of parasite infestations, immune defense, or morphology. We found no indications of such effects of nonrandom assignment, and differences in the change in concentrations of gamma-globulins among treatments thus have to be attributed to differences in die cost of the secondary sexual character. These effects of treatment on gamma-globulins clearly suggest that sexual ornamentation is cosdy in terms of efficiency of the immune defense. Tail-elongated males tended to experience a reduction in immunocompetence, while controls and tail-shortened males showed an increase in gamma-globulin levels. If natural tail length in male barn swallows is costly in terms of immunocompetence, one may wonder why there was no larger increase in immune defense in tail-shortened males than in " 24 / > <.05. SRBC inoculated males whose tail was not manipulated. Previous experimental studies of the barn swallow have shown that tail-shortening resulted in a significant improvement in survival prospects (M0ller and de Lope, 1994), implying that there is a mortality cost of natural tail lengdi among males. Of course, the cost of a long tail may not necessarily be mediated by immune defense, but could be attributed entirely to aerodynamic costs in the breeding areas, during migration, or in the winter quarters. The absence of an increased immune response among males with shortened tails may suggest that there is an optimal level of defense as found among control males, and that any additional investment in immune defense does not result in an improvement in the ability to cope with pathogens. Immunocompetence and differential sexual signaling Models of reliable signaling assume that signals have differential costs widi respect to male phenotypic quality (Grafen, 1990a,b; Heywood, 1989; Iwasa et al., 1991; Zahavi, 1977). Such differential costs should be particularly obvious among individuals that have received an experimentally exaggerated signal, because the costs of the signal should become prohibitively high in the presence of an exaggerated version of the signal. This result was exacdy what was found in the present study: among males with elongated tails there was a significant, positive association between change in concentration of gamma-globulins and original tail length. The increase in the absolute cost imposed by a 2 cm tail elongation was similar for all males, and even larger for originally long Pre-manlputatlon tad length (mm) Figure 4 Relationship between change of gamma-globulin levels and premanipulation tail length in SRBC inoculated males that had their outermost tail feathers experimentally elongated by 2 cm (r =.51, p =.01, n = 24). ~ U

6 232 Behavioral Ecology Vol. 7 No. 2 tailed males if the cost of the ornament increases disprortionately with its size. Hence, the positive correlation between change in gamma-globulin levels and original tail length is consistent with the hypothesis that the cost of a long tail is lower for males with naturally long tails than for short-tailed males. This result is also consistent with previous experiments on barn swallows based on tail length manipulations or manipulations of testosterone plasma levels, demonstrating that naturally long-tailed males experienced lower costs of experimental perturbations than naturally short-tailed males (Meller and de Lope, 1994; Saino et al., 1995). However, such a relationship was absent among unmanipulated males and among males of the three other experimental groups. In conclusion, tail manipulation of male barn swallows in combination with SRBC injection revealed that tail length is cosdy and that estimates of immunocompetence are decreased by tail elongation. Furthermore, males with naturally long tails were better able to cope with tail elongation without compromising dieir ability to raise an immune response to a novel challenge to the immune system. Our study was supported by grants from Consiglio Nazionale delle Ricerche (to N.S.) and the Danish Natural Science Research Council (to A.P.M.). REFERENCES Andersson M, Sexual selecnon. Princeton, New Jersey: Princeton University Press. DeVaneyJA, Augustine PC, Correlation of estimated and actual northern fowl mite populations with the evolution of specific antibody to a low molecular weight polypeptide in the sera of infested hens. Poult Sc 67: Evans M, Thomas ALR, The aerodynamic and mechanical consequences of elongated tails in the scarlet-tufted malachite sunbird: measuring the cost of a handicap. Anim Behav 43: Folatad A, Karter AJ, Parasites, bright males and the immunocompetence handicap. Am Nat 139: Crafen A, 1990a. Biological signals as handicaps. J Theor Biol 144: Grafen A, 1990b. Sexual selection unhandicapped by the Fuher process. J Theor Biol 144: Grossman CJ, Interactions between the gonadal steriods and the immune system. Science 227: Harrison GJ, Harrison LR, Clinical avian medicine and surgery. London: W. B. Saunders. Hawkey CM, Dennet TB, A colour adas of comparative veterinary haematology. Ipswich, U.K.: Wolfe Publishing Ltd. Heywood JS, Sexual selection by the handicap principle. Evolution 43: Iwasa Y, Pomiankowski A, Nee S, The evolution of costly mate preferences. II. The "handicap" principle. Evolution 45: Lochmiller RL, Vestrey MR, Boren JC, Relationship between protein nutritional status and immunocompetence in northern bobwhite chicks. Auk 0: Magnhagen C, Predation risk as a cost of reproduction. Trends Ecol Evol 6: M0ller AP, Female choice selects for male sexual tail ornaments in the monogamous swallow. Nature 332: Meller AP, Effects of an haematophagous mite on the barn swallow (Hirundo rustica): a test of the Hamilton and Zuk hypothesis. Evolution 44: M0ller AP, Parasites, sexual ornaments and mate choice in the barn swallow. In: Bird-parasite interactions: ecology, evolution and behaviour (Love J, Zuk M, eds). Oxford: Oxford University Press; M0ller AP, The bam swallow and sexual selection. Oxford: Oxford University Press. M0ller AP, de Lope F, Differential costs of a secondary sexual character an experimental test of the handicap principle. Evolution 48: M0ller AP, Dufva R., Erritz0e J Host immune defence and sexual selection in birds. Evolution (in press). Nur N, Haxson O, Phenotypic plasticity and the handicap principle. J Theor Biol 0: Saino N, M0ller AP, Secondary sexual characters, parasites and testosterone in the barn swallow Hirundo rusttca. Anim Behav 48: Saino N, M0ller AP, Bolzern AM, Testosterone effects on the immune system and parasite infestations in the barn swallow (Hirundo rustica): an experimental test of the immunocompetence hypothesis. Behav Ecol 6: Sokal RR, Rohlf FJ, Biometry. San Francisco: W. H. Freeman. Soulsby EJL, Immune responses in parasitic infections: immunology, immunopatology, and immunoprophylaxis, vol. IV. Boca Raton, Florida: CRC Press. Svensson L, Identification guide to European passerines. Stockholm: Naturhistoriska Riksmuseet. Tsiagbe VK, Cook ME, Harper AE, Sunde ML, Enhanced immune responses in broiler chicks fed methionine-supplemented diets. Poult Sci 66: Zahavi A, The cost of honesty (further remarks on the handicap principle). J Theor Biol 67: