Ethology Ecology & Evolution 16: 315-328, 2004

Do blood parasites affect reproductive performance in male red bishops (Euplectes orix)? A test of the Hamilton-Zuk hypothesis
R. EDLER, G.M. KLUMP and T.W.P. FRIEDL
1

Carl von Ossietzky Universitt Oldenburg, Institut fr Biologie und Umweltwissenschaften, AG Zoophysiologie & Verhalten, Carl von Ossietzky Str. 9-11, Postfach 2503, D-26111 Oldenburg, Germany
Received 14 June 2004, accepted 27 October 2004

According to the Hamilton-Zuk hypothesis of parasite-mediated sexual selection the expression of male secondary sexual characters as well as health and condition should be negatively affected by parasites. Over five breeding seasons we investigated the relationship between prevalence and intensity of blood parasites and reproductive performance as well as health state in male red bishops (Euplectes orix), a highly polygynous weaverbird species common in southern Africa. Neither prevalence nor intensity of infections with Plasmodium sp., the only blood parasite found in this study, were related to two important measures of male reproductive performance, i.e. the number of nests built (an intrasexually selected trait which is the most important determinant of male mating success) and the ability to establish a territory. In addition, parasite prevalence and intensity were not affected by season, time within season, age or body condition. These results do not support predictions derived from the Hamilton-Zuk hypothesis of parasite-mediated sexual selection. However, our data indicate that high stress levels as assessed by the heterophile/lymphocyte ratio might increase parasite susceptibility.
KEY WORDS:

Hamilton-Zuk hypothesis, blood parasites, Plasmodium, weaverbird, intrasexual selection, number of nests, health state, heterophile/ lymphocyte ratio.

Introduction . . . . . . . Methods . . . . . . . . Results . . . . . . . . Frequency and intensity of parasitism Parasitism and number of nests built Parasitism and territoriality . . Discussion . . . . . . . Acknowledgements . . . . . References . . . . . . . .

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1 Author for correspondence: Dr Thomas W.P. Friedl (E-mail: Thomas.Friedl@unioldenburg.de).

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INTRODUCTION

HAMILTON & ZUK (1982) proposed that male secondary sex characters could signal heritable resistance against parasites and therefore females should prefer highly ornamented mates to obtain genes for parasite resistance for their offspring. Originally developed for plumage brightness, the Hamilton-Zuk hypothesis for parasite-mediated sexual selection has subsequently been applied to all sexually selected traits. Several studies have provided evidence in support of the Hamilton-Zuk hypothesis by demonstrating a negative relationship between parasite prevalence or intensity and the expression of sexually selected traits such as plumage brightness (e.g. SUNDBERG 1995, MERIL et al. 1999, HRAK et al. 2002, DOUCET & MONTGOMERIE 2003), tail length (e.g. SAINO & MLLER 1994), song repertoire (e.g. BUCHANAN et al. 1999), or display rates (e.g. CLAYTON 1990). However, as pointed out by HILLGARTH & WINGFIELD (1997), to be considered as convincing evidence for the Hamilton-Zuk hypothesis, it has to be demonstrated that parasitism not only negatively affects the expression of the sexually selected trait but also reproductive success and survival (or at least body condition) of the host (in addition to parasite resistance being heritable). While some studies have shown that non-parasitized males have a higher mating success than parasitized ones, other studies failed to find any effect of parasites on mating success (reviewed by CLAYTON 1991, ZUK 1992, HILLGARTH & WINGFIELD 1997). Up to now there are only few convincing examples showing unequivocal evidence for parasite-mediated sexual selection (see for example MLLER 1994). In several studies on birds, haematozoan blood parasites have been used to test the different predictions of the Hamilton-Zuk hypothesis. With the exception of microfilariae, most blood parasites are protozoa such as Plasmodium, Haemoproteus, Leukozytozoon, and Trypanosoma, which live in tissues and in the peripheral blood system of their hosts and consume a variety of host metabolites as well as haemoglobin. Most haematozoan parasites have a worldwide distribution (CAMPBELL 1995) and are known to exploit the avifauna in every region they occur. The pathogenicity of blood parasites varies among hosts and parasites. However, detrimental effects of avian blood parasites on the condition and/or reproductive success of their hosts have been reported for several bird species (e.g. RTTI et al. 1993, SUNDBERG 1995, DUFVA 1996, HAKKARAINEN et al. 1998, OTS & HRAK 1998, FIGUEROLA et al. 1999, MERIL & ANDERSSON 1999, DAWSON & BORTOLOTTI 2000), indicating that blood parasites can be suitable types of parasites for tests of the Hamilton-Zuk hypothesis. In this study we examine the relationship between blood parasite infection, reproductive performance, and health status in males of a free living colony of red bishops (Euplectes orix). The red bishop is a colony-breeding polygynous weaverbird common in Southern Africa. Territorial male red bishops, showing a conspicuous red and black breeding plumage, establish and defend small territories, where they construct several nests during a breeding season in an effort to attract females. Male red bishops are characterized by a delayed plumage maturation and moult into the breeding plumage only from their second year on (SKEAD 1956, FRIEDL 2004). While one-year-old males in eclipse plumage occasionally built nests and courted females, they never were able to establish and defend a breeding territory for more than a week (FRIEDL & KLUMP 1999, FRIEDL 2004). In breeding colonies, that are typically located in reedbeds or bulrush stands around water, nesting sites

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are limited. Thus, not every adult male in full breeding plumage can obtain a territory and several non-territorial males (i.e. floaters) were observed at the breeding colony in all study seasons. When a female enters a territory and starts to inspect nests, the male performs a courtship display until the female either flies off or permits copulation. Males provide no parental care, and the territories provide no food or any other resource important to the females with the exception of the nests. The only factor found to be related to male mating success is the number of nests a male builds in his territory which explains up to 85% of the variance in reproductive success ( F RIEDL & K LUMP 1999), while both courtship display and plumage characteristics seem to be unimportant (see F RIEDL & K LUMP 2002, L AWES et al. 2002). The number of nests built is also a good indicator of male quality, since territorial males that survived and established a territory in the following season (thereby demonstrating good quality) built more nests than males that were not able to establish a territory in the following season ( F RIEDL & K LUMP 1999). Males that build many nests, however, do not have a disproportional high mating success, i.e. the females do not settle preferentially in territories with many nests but rather settle randomly ( F RIEDL & K LUMP 2000, L AWES et al. 2002). The reason for this seems to be an increased predation risk if too many females breed within a males territory, offsetting potential advantages of obtaining good genes for the offspring when preferentially settling in territories of males that built many nests (see FRIEDL & K LUMP 2000). By settling randomly, females have a good chance of getting a high-quality mate without incurring any costs of mate-searching behaviour, since they are more likely to end up in the territory of a high-quality male which provides many nests than in the territory of a low-quality male which provides only few nests (FRIEDL & KLUMP 2000). Thus, there is strong sexual selection for male red bishops to build as many nests as possible without apparent female choice, since in a mating system with random female settlement male mating success will be directly related to the number of nests built, which per se will mainly be determined through male-male competition ( FRIEDL & K LUMP 2000, LAWES et al. 2002). The number of nests built is thus a sexually selected trait derived from intrasexual selection in form of male-male competition. Until now, tests of the Hamilton-Zuk hypothesis have been conducted mainly for species with mating systems in which the trait under consideration has been sexually selected through female choice (see above). The mating system of the red bishop offers the opportunity to test whether the Hamilton-Zuk hypothesis can also apply to systems in which the trait (i.e. the number of nests built) has been sexually selected through male-male competition. If the expression of the sexually selected trait number of nests built reflects a trade-off between sexual selection pressures and resistance to pathogens (here blood parasites) as proposed by the HamiltonZuk hypothesis, a negative relationship between parasite prevalence or intensity and the number of nests built would be expected. Furthermore, if blood parasites have detrimental effects on reproductive performance, non-parasitized or less parasitized individuals should also have a higher probability of establishing a territory than their more parasitized competitors. Finally, we would predict that parasites have detrimental effects on health (as assessed by leukocyte counts) and/or body condition of adult males.

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METHODS

Study site The study took place in five consecutive breeding seasons (approx. beginning of October until end of March) between 1994 and 1999. The study site was a small dam (approx. 250 m2) in the Addo Elephant National Park, Eastern Cape Province, South Africa (3326 S; 2545 E), where a red bishop population was breeding in the surrounding vegetation mainly consisting of bulrush (Typha capensis) and common reed (Phragmites australis). The study was conducted under licence from the Provincial Administration of the Cape of Good Hope, Chief Directorate Nature and Environmental Conservation (Licence numbers: 653/94, 144/95, 92/96, 1/97) and with permission from the South African National Parks Board. A detailed description of red bishop breeding behavior is given in FRIEDL (2004).

Field methods Nest counts were conducted daily or every second day to register new nests built by the territory holders. These new nests were marked with numbered yellow plastic tags attached to a reed or bulrush stem close to the nest. All tagged nests were checked at least every second day to collect data on nest acceptance, clutch size, number of hatchlings and number of fledglings. Throughout the breeding season, detailed observations of territorial behavior and aggressive interactions between territorial males provided information on the location of territories and the identity of territory holders. Adult red bishops were caught in baited walk-in traps or with mist nets set up close to the border of the colony. We measured tarsus length and body weight of most captured individuals. Each bird was leg-banded with a unique combination of four colored plastic rings. In addition, every bird received a numbered stainless steel ring provided by the South African Bird Ringing Unit. More details of the study site and general field methods are provided in FRIEDL & KLUMP (1999).

Blood smears For the analyses of infection with blood parasites and to obtain the haemogram a drop of blood was taken from every caught male by puncturing the brachial vein with a sterile 25gauge needle. Blood was collected with a heparinisized microcapillary tube and smeared on a microscopic slide. The smears were air-dried and stored in boxes. If a male bird was caught more than once in a season, one of the obtained smears was chosen randomly; therefore only one smear per bird and season was used for the analyses. We only used blood smears from males in breeding plumage, indicating that a male is old enough to compete for a territory (see above). In the season 1997-1998 blood smears were collected from territorial but not from non-territorial males. A total of 131 blood smears from 98 different individuals caught during the five study seasons was available. For 27 males blood smears were collected in more than one season, with blood smears from two seasons being available for 21 males and blood smears from three seasons being available for 6 males. The person examining the blood smears had no information about the individual birds except for an identification number on the slides.

Haemogram A haemogram (blood cell count) describes the proportion of different leukocytes, which are part of the immune system and thus reflect the health state of an individual. To obtain

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the haemogram air-dried blood smears were stained with Wrights stain and examined with a microscope under 640 magnification. In areas where blood cells formed a monolayer we counted the number of erythrocytes and leukocytes, which where subdivided in lymphocytes, monocytes, basophilic granulocytes, eosinophilic granulocytes and heterophilic granulocytes. We examined neighboring consecutive microscopic fields at a right angle to the direction of the blood smear. When we reached the border of the blood smear, the next field counted was always the one to the right. Thereby we avoided double counts of microscopic fields. Every smear was examined until we counted a total of 100 leukocytes. Based on these counts the heterophile/lymphocyte ratio (HLR) was calculated. To assess the repeatability for our counts, 12 blood smears were randomly chosen and recounted without knowledge of the results of the first count. All relative and absolute counts as well as the HLR showed high and significant repeatability (Spearman rank correlation coefficients, n = 12, all rs > 0.6, all P < 0.05), with repeatabilities for relative lymphocyte counts, relative heterophile counts, and the HLR being particularly high (all rs > 0.9, all P < 0.001). The haemogram can be influenced by various stressors, such as infectious diseases, parasite infection, malnutrition and social-psychical disruption. In particular the HLR responds to those stressors with a progressive increase (OTS & HRAK 1996, BIRKHEAD et al. 1998, HRAK et al. 1998, ILMONEN et al. 2003), and is thus a measurement commonly used to assess stress levels and health state of birds (e.g. GROSS & SIEGEL 1983, GYLSTORFF & GRIMM 1998, MAXWELL & ROBERTSON 1998, SAMOUR 2000). Since stress is known to suppress immune functions (SAPOLSKY 1992, APANIUS 1998) a high HLR is indicative of immunosuppressive conditions with a concomitant increase in susceptibility to infections (SIEGEL 1995, OTS et al. 1998).

Blood parasites While obtaining the haemogram, the stained blood smears were also scanned for the presence of the blood parasites Trypanosoma and Leucocytozoon. We examined the slides for the presence of Haemoproteus and Plasmodium under oil immersion at 1000 magnification for 20 min, scanning approximately 200 microscopic fields during that period of time. Individuals were judged as parasitized with a certain type of parasite when that parasite was detected in the smear, regardless of the number in which it occurred. Intensity of parasitism is the number of parasites of a certain type we found in twenty minutes of scanning.

Data analyses and statistics We used general linear models (GLM) to test whether occurrence or intensity of bloodparasite infections were related to reproductive performance and/or health status of male red bishops. To assure statistical independency, every male entered the analyses only once (selected at random). When we investigated blood parasite prevalence, we performed GLMs with binomial errors and a logit link, since the response variable (whether or not blood parasites were found in a smear) is binary. When we investigated blood-parasite intensity, we used GLMs with the number of parasites found in a blood smear as the continuous response variable. In all analyses we included season as a factor and time within season at which a blood sample was collected (hereafter only time within season) as covariate, since these variables might influence blood-parasite prevalence and intensity. Time within season was calculated as number of days after the day on which the first egg was laid within the study colony (day 1). There were five cases in which a blood sample was collected before the first egg was laid within the colony (never more than 7 days before the first egg was laid); in these cases time within season was set to one. Besides season and time within season, we included HLR, body condition index (BCI) and male minimum age as covariates to investigate whether parasite prevalence and/or intensity were affected by these variables. The BCI was calculated as the residual of the regression of body weight against tarsus length. Male minimum age was used

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because most males were caught for the first time as adults wearing full breeding plumage; thus their exact age could not be determined. Since male red bishops moult into breeding plumage in their second year (SKEAD 1956, FRIEDL 2004), these males were at least two years old when caught for the first time. Therefore, minimum age in the year of blood sample collection was 2 years plus the number of years between first capture and blood sample collection. Males that were caught for the first time as adults in nonbreeding plumage were oneyear old at the time of first capture. For these males and males that were marked as nestlings we thus knew the age and included the known exact age (instead of minimum age) as covariate into the model. According to the Hamilton-Zuk hypothesis, there should be a negative relationship between the sexually selected trait number of nests built and parasite prevalence or intensity (see above). Thus, we would expect that males that built many nests are characterised by a lower degree of parasitism than males that built only a few nests. To test this, we performed GLMs with either parasite prevalence (i.e. whether or not blood parasites were found in a smear) or parasite intensity (number of blood parasites found in a smear) as the response variables. Besides the number of nests built the models included season (as factor) and time within season, minimum age, BCI and HLR as covariates. Generally, the Hamilton-Zuk hypothesis predicts that parasites have detrimental effects on reproductive performance; therefore we would expect non-parasitized or less parasitized individuals to have a higher probability of establishing a territory within the study colony than their more parasitized competitors (see above). This was tested by performing GLMs with either parasite prevalence or parasite intensity as response variables, including territoriality (i.e. whether or not a male was able to establish a territory in the season the blood smear was collected) as factor. Since both territorial and non-territorial males entered the analyses, the data set was enlarged compared to the analyses with number of nests built as predictor variable described above. Again, the models also included season (as factor) and time within season, minimum age, BCI and HLR as covariates. The prediction of the Hamilton-Zuk hypothesis that parasites have detrimental effects on health or body condition of males was investigated by including both the HLR (as measure of stress and general health) and BCI as covariates in all analyses reported here. Thereby we could test whether health or body condition were directly related to parasite prevalence and/or intensity.

RESULTS

Frequency and intensity of parasitism The only blood parasite found in the study was Plasmodium sp., which was identified in 54 (i.e. 41.2%) of the 131 blood smears investigated (see Table 1). The mean number of Plasmodium counted in the 54 blood smears was 24.5 26.5 (range 3 to 141). The repeatability of the intensity of blood parasite infection for individual males between seasons was very high, as shown by the significant positive correlation between the number of Plasmodium counted in blood smears obtained for the same male in different years (Spearman correlation coefficient rs = 0.8, P < 0.001, n = 27). Parasitism and number of nests built A GLM with parasite prevalence (yes or no) as binary response variable, the number of nests built as predictor variable representing reproductive performance,

Blood parasites and sexual selection in red bishops

Table 1.

321

Descriptive statistics for blood parasite prevalence and intensity (number of Plasmodium; median and range) of territorial and non-territorial male red bishops. Season Male status territorial 1994/95 non-territorial 6 4 15; 4-44 Number of nonparasitized males 13 Number of parasitized males 11 Blood parasite intensity 38; 4-141

territorial 1995/96 non-territorial

11 4

6 6

12; 3-49 15; 6-63

territorial 1996/97 non-territorial

12 6

6 4

8; 5-24 11; 3-32

territorial 1997/98 non-territorial

4 *

1 *

12 *

territorial 1998/99 non-territorial

15 6

12 4

18; 5-35 25; 4-117

territorial All seasons non-territorial

55 22

36 18

17; 3-141 13; 3-117

* In the season 1997/98 no blood smears from non-territorial males were collected.

and season, time within season, minimum age, BCI, and HLR as additional independent predictor variables, revealed no significant effect of any of the variables investigated. In addition, a GLM with parasite intensity (number of Plasmodium) as dependent variable and the same set of independent variables as above again showed that the sexually selected trait number of nests built was not significantly related to parasite intensity, with the effect size (as assessed by the Pearson correlation coefficient) being 0.109. However, as shown in Table 2, there was a significant relationship between HLR and blood parasite intensity. A high HLR (which indicates high stress level) is related to a greater number of Plasmodium (see Fig. 1), suggesting an increase in susceptibility against parasites associated with higher stress levels. It has to be noted, however, that the significant effect of HLR was solely due to the one male with the by far highest blood parasite intensity. Removing this single data point from the analysis yielded a non-significant full model (P =

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Table 2.

Results of a GLM with blood parasite intensity (number of Plasmodium) as response variable, season as factor, and number of nests built, time within season, minimum age, body condition index (BCI), and heterophil/lymphocyte ratio (HLR) as covariates. Every male entered the analyses only once to assure statistical independency. Parameter Modela Intercept Season Time within season Minimum age BCI HLR Number of nests built Error Total
a

Sum of squares (Type III) 10147.85 38.78 1729.90 496.25 5.99 213.38 1872.65 0.04 18543.07 35655.00

df 9 1 4 1 1 1 1 1 49 59

F 2.980 0.102 1.143 1.311 0.016 0.564 4.948 0.000

P 0.007 0.750 0.348 0.258 0.900 0.456 0.031 0.992

Adjusted R2 = 0.235.

120

Parasite intensity

r = 0.526 P < 0.001 n = 59

80

40

HLR
Fig. 1. Relationship between the heterophile/lymphocyte ratio (HLR) and blood parasite intensity (number of Plasmodium). The arrow indicates the single data point on which the significance of the whole model was based (see text).

0.229, adjusted R2 = 0.055). This indicates that an effect of HLR on blood parasite intensity is only found at very high stress levels; that is, only very high stress levels

Blood parasites and sexual selection in red bishops

Table 3.

323

Results of a GLM with blood parasite intensity (number of Plasmodium) as response variable, season and territoriality (territory owner yes or no) as factor, and time within season, minimum age, body condition index (BCI), and heterophil/lymphocyte ratio (HLR) as covariates. Every male entered the analyses only once to assure statistical independency. Sum of squares (Type III) 5042.87 352.80 2950.57 214.95 672.57 1.79 193.00 1343.55 41028.58 55461.00

Parameter Modela Intercept Season Territoriality Time within season Minimum age BCI HLR Error Total
a

df 9 1 4 1 1 1 1 1 78 88

F 1.065 0.671 1.402 0.409 1.279 0.003 0.367 2.554

P 0.398 0.415 0.241 0.525 0.262 0.954 0.546 0.114

Adjusted R2 = 0.012.

seem to increase the susceptibility of a male to parasitism. There were no significant effects of season, time within season, minimum age or BCI. Parasitism and territoriality We repeated the analyses described above with territoriality (yes or no) instead of the number of nests built as independent variable reflecting reproductive performance. Sample size was increased compared to the analyses above because all males in breeding plumage present at the study colony with and without a territory were included, while in the previous analyses only males were included that were successful in establishing a territory. Neither a GLM with parasite prevalence as binary response variable nor a GLM with parasite intensity as continuous response variable revealed a significant effect of any of the independent variables included (see Table 3).

DISCUSSION

Our data show that in male red bishops neither blood parasite prevalence nor blood parasite intensity were related to two important measures of male reproductive performance, i.e. the number of nests built and the ability to establish a territory. Thus our results do not lend support for the Hamilton-Zuk hypothesis, which proposes a negative relationship between the sexually selected trait number of nests built and parasitism. In addition, we found no effect of season, time within season, minimum age and body condition on parasite prevalence or intensity. However, our finding that the male with the highest HLR had the highest number

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of blood parasites indicates that high stress levels as assessed by the HLR might increase the susceptibility to blood parasites. The only blood parasite detected in our study was Plasmodium, a haematozoan parasite which causes avian malaria. At least 14 different species of Plasmodium have been described for the avifauna of the sub-Saharan region, with two species (Plasmodium relictum and P. rouxi) reportedly occurring in the red bishop (see BENNETT et al. 1992). Plasmodium is known to be pathogenic in some canaries, penguins, domestic poultry, ducks, pigeons and falcons, while several passerine species are frequently asymptomatic carriers of Plasmodium (CAMPBELL 1995, GYLSTORFF & GRIMM 1998). Thus, the lack of any significant relationship between parasitemia and reproductive performance in male red bishops could be explained by Plasmodium being relatively harmless for the birds. Infections with Plasmodium might not severely harm male red bishops, at least not at the low to medium levels of intensity found in this study. There is also the possibility that we did not detect detrimental effects of Plasmodium in our study, even if the parasite is severely harmful to red bishops. This would be the case if infections rapidly eliminate heavily parasitized individuals from the population, leaving only non-infected individuals or individuals with a chronic or non-virulent level of infection, which are to some degree resistant to reinfection (YORINKS & ATKINSON 2000, VALKIUNAS 2001). Males with an acute infection of avian malaria, on the other hand, would not even appear in the breeding colony, much less compete with other males for breeding opportunities ( YORINKS & ATKINSON 2000, VALKIUNAS 2001). Other variables that could potentially influence the effects of parasitism include seasonal changes (e.g. ALLANDER & SUNDBERG 1997) and age (e.g. HRAK & OTS 1998). However, we found no relationship between time within a breeding season a blood sample was collected and parasite prevalence or intensity (see Results), and parasitism was also unrelated to minimum age of the males (see Results). In some studies food availability has been reported to influence parasite prevalence, with higher susceptibility to parasitism in seasons with low food abundance (e.g. KORPIMKI et al. 1993, ILMONEN et al. 1999, WIEHN et al. 1999). For red bishops, it has been shown that overall breeding activity in a season is correlated with the amount of rainfall, which is necessary to provide enough food resources for the breeding birds (FRIEDL 2002). However, we did not find an effect of season on blood parasite prevalence or intensity despite the fact that the study seasons differed markedly with respect to the amount of rainfall and thus food availability (see FRIEDL 2002). As stated above, our finding that the male with the highest HLR had the highest number of blood parasites suggests that high stress levels might increase parasite susceptibility. It is generally assumed that stress induces immunosuppression (e.g. SAPOLSKY 1992, APANIUS 1998) and several experimental studies on birds revealed that various stressors suppress immune functions (LOCHMILLER et al. 1993, DEERENBERG et al. 1997, BIRKHEAD et al. 1998, SVENSSON et al. 1998, ILMONEN et al. 2003). Reproductive effort itself could also increase the susceptibility to parasitism, as investment into reproduction may limit the energy available for maintenance of the immune system and the clearance of parasites through cell-mediated immunity, both of which have been shown to be energetically costly (reviewed by LOCHMILLER & DEERENBERG 2000). Positive associations between reproductive effort and blood parasite infections have been documented in various bird species (e.g. NORRIS et al. 1994, OTS & HRAK 1996, ALLANDER 1997, OPPLIGER et al. 1997, SIIKAMKI et al.

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1997, NORDLING et al. 1998), supporting the idea that a trade-off exists between reproductive effort and immune function, and, therefore, that immune suppression caused by reproductive effort might be an important mechanism mediating the costs of reproduction. However, in our study we did not find that parasite prevalence or intensity were significantly related to reproductive performance in the red bishop. Using a meta-analytical approach, MLLER et al. (1999) found evidence for an overall negative relationship between parasite load and the expression of secondary sexual characters, with a mean effect size (as assessed by Pearsons correlation coefficients) of 0.123. The effects, however, were much stronger for studies based on host immune responses (mean effect size 0.465) as compared to studies based on parasite loads directly (mean effect size 0.06). The effect size of 0.109 found in our study is similar to the mean effect size of parasite load on the expression of secondary sexual characters reported by MLLER et al. (1999), indicating that our result even if not statistically significant is in accordance with the general findings. As pointed out by MLLER et al. (1999), the many negative (i.e. non-significant) results reported by previous analyses of parasite-mediated sexual selection (including our own) may be explained by relatively benign parasites being investigated; thus it might be better to investigate parasite-mediated sexual selection using measures of host antiparasite defenses rather than data on parasite prevalence or intensity itself. Finally, there is another possible explanation for the fact that we found no association between infection with Plasmodium and male reproductive performance or condition in red bishops. Data on parasite prevalence and intensity obtained by the technique of counting blood smears though widely used in studies testing predictions of the Hamilton-Zuk hypothesis might not be accurate enough to detect small effects. Especially infections with Plasmodium are often low-level infections difficult to detect and quantify in blood smears (BENNETT et al. 1992, VALKIUNAS 2001). In the past few years PCR techniques have been developed that allow the detection of low-level infections with Plasmodium (e.g. CICERON et al. 1999, THAM et al. 1999), and, more recently, PCR-based methods for a quantitative assessment of malaria infections in humans have been established (e.g. SCHOONE et al. 2000). It might well be that tests of the Hamilton-Zuk hypothesis using data obtained with such PCR-based methods which are more accurate and reliable than data obtained by counting parasites in blood smears (e.g. FELDMAN et al. 1995, THAM et al. 1999) reveal effects of blood parasites on reproductive performance which could not be detected by the still widely used but less accurate method of counting parasites in blood smears.

ACKNOWLEDGEMENTS We are grateful to the National Parks Board of South Africa and the Chief Directorate Environmental Affairs of the Department of Economic Affairs, Environment and Tourism of the Eastern Cape Province for permission to conduct this study in the Addo Elephant National Park. We want to thank the whole park staff of the Addo Elephant National Park for continuous support. Many thanks to Nicole Geberzahn and Lars H. Hansen who helped with the field work. Two anonymous reviewers provided helpful comments on the manuscript. T.W.P. Friedl was supported by grants from the DAAD (Deutscher Akademischer Austauschdienst), the Friedrich-Schiedel-Stiftung, the Stifter-Verband, and by a scholarship from the Technical University Munich. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Kl 608/11-1,2 and 13-1,2).

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