Reproductive investment and parasite susceptibility in the Great Tit

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1 Functional Ecology 1997 Reproductive investment and parasite susceptibility in the Great Tit K. ALLANDER* Department of Zoology, Villavägen 9, S Uppsala, Sweden Summary 1. Reproduction and parasite defence are assumed to be costly activities for hosts, and therefore trade-offs might exist between reproduction and parasite defence. 2. Brood sizes were manipulated in a population of Great Tits (Parus major L.) to assess trade-offs between reproduction and parasite defence. Blood samples were taken from males and females during the late nestling phase, and parasite prevalence was compared among parents raising enlarged, reduced and control broods. 3. A higher prevalence of protozoan blood parasites dominated by Haemoproteus majoris was found among parents with enlarged broods, as compared with parents with control and reduced broods, respectively. Brood size manipulation did not affect parasite prevalence between the sexes or between age classes differently. 4. Mean parasite intensity (number of parasites per microscope field) among adult birds only infected with H. majoris was higher for parents with experimentally enlarged broods than controls and reduced broods, respectively. 5. These results support the hypothesis of a trade-off between parasite defence and reproduction. Key-words: Blood parasites, brood size manipulation, Haemoproteus majoris, Parus major, trade-off Functional Ecology (1997) Ecological Society Introduction Life-history theory assumes that reproduction is costly and competes for resources with other costly activities performed by individuals. An individual cannot simultaneously maximize all life-history traits since energy is a limiting resource that has to be optimally allocated among different functions (Williams 1966; Stearns 1992). Thus, investment in current reproduction may be at the expense of future reproduction and/or survival (i.e. there is a cost of reproduction). Parasites draw their resources from the living bodies of other organisms and live some or all of their life on or in their hosts (Price 1980). This means that parasites will utilize host resources, which might otherwise have been used for maintenance, survival and/or reproduction. Therefore, parasites are expected to affect the fitness of their hosts negatively (see Møller, Allander & Dufva 1990; Lehmann 1993 for reviews). Since both reproduction and parasite defence are costly, an animal will have to balance the proportion of available resources channelled into one or the other (Møller 1997). If an animal increases its reproductive effort (for example, by raising a larger * Present address: ETH Zürich, Experimental Ecology ETH-Zentrum NW, CH 8092 Zürich, Switzerland. brood), it may have fewer resources for parasite defence. This will increase its susceptibility to parasites, which may have effects on its future reproduction and/or survival prospects. A trade-off between reproductive effort and the ability to mitigate parasitic infections has recently been demonstrated experimentally in birds where an increased reproductive effort resulted in an increased prevalence of blood parasites in the Great Tit Parus major L. (Norris, Anwar & Read 1994; Richner, Christe & Oppliger 1995). This has also been demonstrated in mammals, where lactating bighorn Ovis canadensis ewes had higher counts of lungworm larvae than non-lactating ewes (Festa-Bianchet 1989). It has been suggested that even if an increased reproductive effort may lower the condition of an individual, condition may be built up rapidly particularly in small birds that have daily accumulation and depletion of energy reserves (Murphy & Haukioja 1986). This suggests that the cost of reproduction will not be due to an increased energy demand only. If, by contrast, an increased reproductive effort leads to a reduced immune function, an increase in parasite intensity will further result in a lower condition. The fact that birds often have relapses of blood parasites long after the end of the breeding season (K. Allander & J. Sundberg, unpublished data) supports 358

2 359 Reproductive investment and parasite susceptibility the notion that the effects of an increased intensity of parasites may operate for a considerable time. Thus, the trade-off between reproductive effort and parasite defence could be one mechanism that operates in the observed patterns of costs of reproduction (reviewed by Lindén & Møller 1989). The aim of this study was to investigate whether experimentally increased reproductive effort (achieved by altering brood size) might affect the prevalence and intensity of avian blood parasites in Great Tits P. major. This particular population of Great Tits has previously been found to be infected by a variety of blood parasites with Haemoproteus majoris being by far the most common, infecting around 75% of the adults depending on year and age of the birds (Allander & Bennett 1994). The paper also compares results from similar experiments of trade-offs between reproductive effort and parasite defence in other Great Tit populations exposed to different environments and parasites (i.e. Norris et al. 1994; Richner et al. 1995). Methods STUDY SPECIES AND STUDY AREA The study was carried out in 1993 using a population of Great Tits breeding on the island of Gotland in the Baltic, SE Sweden (5710'N, 1820'E). The study area contains about 950 nestboxes distributed among 13 different plots. Twelve plots consist of deciduous woodland with ash (Fraxinus excelsior), oak (Quercus robur), birch (Betula spp.), hazel (Corylus avellana) and hawthorn (Crataegus spp.), whereas one plot is mainly coniferous forest, dominated by pine (Pinus sylvestris) with some birch. The Great Tit is a small (c. 20 g), mainly insectivorous passerine bird, resident in the study area, which breeds readily in nestboxes. All nestboxes were cleaned before the breeding season by removing nesting material from the previous year. Nestboxes were regularly inspected to determine the date of laying of the first egg, clutch size, hatching date (of first egg), number of hatchlings and number of fledglings. Only data from first clutches were used. Nestlings were banded and their tarsus length, body mass and wing length measured at an age of 13 days (hatching day = day 0). In most cases tarsi are fully grown and body mass has reached an asymptote when nestlings are 13- days old (Perrins 1979). During the nestling period when nestlings were 10 to 16-days old, adult birds were trapped in the nestbox (n = 335), banded or identified from bands already present, aged and measured. Birds were classified as either 1-year old or 2 years old using the criteria described by Svensson (1984). At the same time, blood samples were taken from all breeding adults from the brachial vein of the left wing in 20-µl heparinized micropipettes and smeared on individually marked microscope slides, air-dried, fixed in 100% methanol and stained with Giemsa stain. Blood parasites, which are protozoan parasites transmitted by dipteran vectors, were identified and quantified by counting the number of parasites per 100 fields under a 100 oil immersion objective by Gordon F. Bennett who had no knowledge about the treatment to which the individual birds were subjected. Prevalence of blood parasites is defined as the proportion of infected birds in a sample, while intensity of infection of individual hosts is defined as the number of parasites per microscope field in a blood smear. Allander & Bennett (1994) reported a very high repeatability (0 97) of parasite intensity within smears. BROOD MANIPULATION EXPERIMENT Brood size manipulations were performed on the day after hatching. Broods in the enlargement group received nestlings of the same age as those already present in the box. Nestlings added (increased reproductive effort) to broods were transferred from other nests (reduced reproductive effort) randomly chosen among broods with the same hatching date within the study area. Such nestling transfers typically have no effect on nestling survival or growth (cf. Alatalo & Lundberg 1989). Broods with eight young received two extra nestlings and broods with nine young three extra nestlings in order to increase the burden proportionally. Since there was no significant difference in prevalence of parasites between the broodenlarged groups (i.e. 2 (n = 7) and 3 (n = 28) nestlings added; males, χ 2 = 1 83, df = 1, P = 0 17; females, χ 2 = 0 07, df = 1, P = 0 79) and the brood-reduced groups (i.e. 2 (n = 8) and 3 (n = 29) nestling removed; males, χ 2 = 1 42, df = 1, P = 0 23; females, χ 2 = 0 10, df = 1, P = 0 76), the two enlarged groups were pooled as were the two reduced groups. The control group contained unmanipulated broods whose laying dates were within the same time span as those of the experimental group. There was no significant difference in natural clutch size between enlarged, control and reduced broods (enlarged, n = 35, mean = 8 84, SE = 0 28; controls, n = 115, mean = 8 47, SE = 0 14; reduced, n = 37, mean = 8 84, SE = 0 20; ANOVA, F 2,186 = 1 55, P = 0 21). Furthermore, no statistically significant differences were found between experimental groups in age class distribution (χ 2 = 0 18, df = 2, P = 0 91), laying date (enlarged, mean = 32 4, SE = 0 49; control, mean = 33 6, SE = 0 32; reduced, mean = 32 5, SE = 0 55, ANOVA, F 2,182 = 2 28, P = 0,11), and body size (tarsus length) in both females and males (males, ANOVA, F 2,163 = 1 04, P = 0 36; females, ANOVA, F 2,169 = 0 52, P = 0 59). DATA ANALYSIS The effects of sex, age (1-year old, 2-years old) and brood size manipulation (decreased, unmanipulated,

3 360 K. Allander enlarged) on prevalence of parasites (infected vs not infected) in the population of adults was tested with log-linear analysis under the procedure CATMOD in SAS (SAS Institute Inc. 1988). It tests for associations between categorical variables and can be regarded as a counterpart to analysis of variance (Sokal & Rohlf 1981). It was tested whether males and females responded differently to brood size manipulation in terms of parasite prevalence and if age had any effect on the experimental outcome. In these analyses a hierarchy of models was used starting with the most complex including one three-way interaction and all three possible two-way interactions. Progressively simpler models were then fitted to the data in an attempt to drop all non-significant interactions. To determine the significance of a given variable, the difference in deviance between a model with and without the variable of interest was calculated (Sokal & Rohlf 1981). If the three-way interaction was not significant (i.e. if the association between any two variables did not depend upon the third variable), it was dropped from further analysis. The next step was to drop the nonsignificant two-way interactions in further analysis until the significant interactions were identified. Results Fig. 1. Prevalence of blood parasites of adult Great Tits during the nestling period in relation to brood size manipulation and sex (a), and for sexes combined (b). Numbers refer to number of individuals sampled. The prevalence of different species of blood parasites found in adults from all unmanipulated nests were Haemoproteus majoris 50 9%, Hepatozoon parus 14 5% and Plasmodium vaughani 5 5%. One bird in the enlarged group was also found to be infected with Trypanosoma avium. The distribution of different parasite species in this study corresponded with the results in Allander & Bennett (1994). The brood manipulation experiment resulted in significantly more fledglings in the enlarged group than in the controls and reduced group, respectively (enlarged, n = 35, mean = 10 03, SE = 0 51; controls, n = 115, mean = 7 63, SE = 0 19; reduced, n = 37, mean = 5 62, SE = 0 20; ANOVA, F 2,186 = 37 26, P < 0 001). In an observational study, Allander & Bennett (1994) found no sex difference in terms of parasite prevalence in the same population of Great Tits over the course of three years. In the present study, the prevalence of blood parasites within the control group again was similar in both sexes, according to blood samples taken during the nestling provisioning period (χ 2 = 0 09, df = 1, P = 0 77). However, since both Norris et al. (1994) and Richner et al. (1995) reported a difference in prevalence of blood parasites between males and females, both in an unmanipulated situation and in response to brood size manipulation, sex was included in the analysis in the present study. Figure 1 shows the prevalence of blood parasites in relation to the brood size manipulation, both for sexes separately (Fig. 1a) and for both sexes combined (Fig. 1b). Loglinear analysis showed no significant three-way interaction between sex, manipulation and parasite prevalence (χ 2 = 1 19, df = 2, P > 0 50) and no significant interaction between sex and parasite prevalence (χ 2 = 0 07, df = 1, P > 0 70). However, there was a significant interaction between manipulation and parasite prevalence (χ 2 = 10 00, df = 2, P < 0 01). This means that while parasite prevalence was related to manipulation, the response to the manipulation was similar in males and females. Allander & Bennett (1994) previously demonstrated a difference in prevalence of blood parasites between age classes, with older birds ( 2-years old) having a higher prevalence than 1-year-old individuals. Conceivably, older individuals could by chance have become over-represented in the brood-enlarged group. However, there was no difference in the age class distribution between the different treatments (see Methods). Another possibility is that different age classes may respond differently to manipulation. Older individuals may be more capable than young individuals to depress an infection through acquired

4 361 Reproductive investment and parasite susceptibility Fig. 2. Prevalence of blood parasites from the nestling period in adult Great Tits in relation to brood size manipulation for different age classes. Numbers refer to number of individuals sampled. immunity. Thus, young birds may represent a larger fraction of birds with parasites in the increased group as a result of their lower ability to resist or fight a parasite infection. To check this, the prevalence of blood parasites in relation to age and manipulation was examined, and the results are shown in Fig. 2, which reveals that birds of the two age classes responded similarly to the brood size manipulation. The inconsistency in n-values between Fig. 1(a), (b) and Fig. 2 was due to the fact that five birds were not aged. Loglinear analysis revealed a statistically non-significant three-way interaction (age class manipulation prevalence; χ 2 = 3 74, df = 2, 0 10 < P < 0 20) and a statistically non-significant two-way interaction between age class and prevalence (χ 2 = 1 89, df = 1, P > 0 30), but a significant interaction between brood size manipulation and parasite prevalence (χ 2 = 10 15, df = 2, P < 0 01). Thus, there was no statistical evidence for any age-related differences with respect to the response to the brood size manipulations. To determine whether parasite intensity was associated with reproductive effort, only individuals exclusively infected with H. majoris were examined. Allander & Bennett (1994) previously demonstrated that older birds had a lower parasite intensity than 1- year-old birds. The distribution of parasite intensity was far from normal even when it was log-transformed. Consequently, a two-way ANOVA using age class and brood size manipulation as independent variables was not possible. Therefore, parasite intensity was analysed in relation to reproductive effort within age classes using the Kruskall Wallis test as a base for the ordered heterogeneity test (Rice & Gaines 1994) because of the expected order of means across experimental groups. There was a significant positive relationship between parasite intensity and reproductive effort in 1-year-old birds (Fig. 3; ordered heterogeneity test; r s P c = 0 91, n = 82, k = 3, P < 0 005) as well as in 2-year old birds (Fig. 3; ordered heterogeneity test; r s P c = 0 72, n = 62, k = 3, P = 0 05). Feeding rate was not measured, but as an indirect measurement of reproductive effort the summed brood mass may be used because it is likely to correlate with the amount of food brought to the brood by the parents. Figure 4(a) shows the increase in summed brood mass from day of manipulation to the day of measurement of nestlings (13-days old) in relation to brood size manipulation, assuming a mean nestling mass of 2 3 g at the age of one day (adopted from Schifferli 1973: Table 1). Enlarged broods had a larger summed brood mass increase than control and decreased broods, respectively (ANOVA F 2,181 = 24 02, P < 0 001). Thus, the results indicate that the brood size manipulation probably made the parents to work harder. However, mean nestling mass was negatively related to manipulation (Fig. 4b). Nestlings from decreased broods had on average a larger body mass than nestlings from controls and enlarged broods, respectively (ANOVA F 2,181 = 7 07, P = 0 001). Discussion This study demonstrated that an increase in brood size resulted in an increase in the prevalence of blood parasites in both sexes of the Great Tit. Parents with enlarged broods had a higher prevalence of blood parasites than parents with control and reduced broods, respectively. No statistically significant difference was found between the sexes in prevalence of parasites in relation to brood size manipulation, which contrasts with the results reported by Richner et al. (1995) and Norris et al. (1994); nor was any significant effect of age found. There was a positive relationship between reproductive effort and parasite intensity. Young birds seemed to be more affected by the brood size manipulation, i.e. had a higher mean parasite intensity than Fig. 3. Mean intensity of infection (number of parasites per microscope field) in relation to brood size manipulation and age classes among Great Tits only infected with Haemoproteus majoris. Numbers refer to number of individuals sampled. Bars are SE.

5 362 K. Allander Fig. 4. (a) Summed brood mass increase from manipulation day (1-day-old nestlings) to day of measurement (13-days old) and (b) mean nestling mass in relation to brood size manipulation. Numbers refer to number of broods. Bars are SE. older birds, which may indicate an effect of acquired immunity to parasites in older birds. Furthermore, as indirect evidence that parents responded to manipulation with elevated feeding rates (i.e. increased parental effort), the mean brood mass increase was larger in enlarged broods than in controls and decreased broods, respectively. A relapse of a chronic latent infection of parasites during the breeding season can occur as an effect of physiological and environmental stress (Atkinson & van Riper 1991). The release of sex hormones has been proposed as having a suppressive effect on the immune system, generating physiological stress (Folstad & Karter 1992), or alternatively parasites in the tissues may become activated by such hormones. Chernin (1952) showed in the domestic duck (Anas plathyrhyncos) that a relapse of a chronic latent infection took place when daylength in winter was altered to that prevailing during the breeding season. Environmental stress may increase costs such as time and energy during breeding, for example, as a result of nest building, courtship feeding and mate-guarding activities. Thus, both hormones and the inevitably increased activity may cause a relapse of latent infection during breeding. An additional reason for the increased prevalence of blood parasites during breeding is that host breeding coincides with a maximum abundance of dipteran vectors, which need a blood meal to reproduce and in that process transmit parasites. Therefore, the peak in prevalence of parasites in the host population during the breeding period may in part be due to newly acquired infections. Thus, both increased reproductive stress and vector activity potentially influence the dynamics of prevalence of blood parasites in a host population. Possible explanations for the positive relationship between brood size manipulation and prevalence of parasites may thus be increased stress due to increased feeding rate and/or increased exposure to vector population or less time for antiparasite behaviours as argued both by Norris et al. (1994) and Richner et al. (1995). However, in this study, where H. majoris is the most frequent parasite in adult hosts, this latter explanation seems unlikely, since the incubation period (which is the time from injection by a vector to a patent parasitaemia could be detected) for H. majoris and Plasmodium sp. is days (Garnham 1966; Desser & Bennett 1993), which was approximately the time gap between day of manipulation and blood sampling day. Thus, new infections acquired during this time will be almost impossible to detect. This study demonstrates a trade-off between reproductive effort and the ability to resist parasite infections, as also shown recently in the same species by Norris et al. (1994) and Richner et al. (1995). This trade-off is similar in males and females, which contrasts with the results of other studies of the Great Tit. Gustafsson et al. (1994) in a brood size manipulation experiment with the Collared Flycatcher (Ficedula albicollis) showed that the effect of experimental alteration of brood size in the current year on reproductive performance in a subsequent year was affected by the degree to which individuals were parasitized by blood parasites. Møller (1993) documented a cost of reproduction imposed by parasites within a breeding season. He found that Barn Swallows Hirundo rustica that were subjected to high mite loads and increased brood size in their first broods laid fewer, smaller and later second clutches. Richner et al. (1995) suggested that the higher prevalence of blood parasites observed in male Great Tits with enlarged broods could be explained by their elevated feeding rates. This was supported by the fact that females neither responded to brood enlargement with increased feeding rate nor suffered a higher prevalence of blood parasites. Furthermore, parents with reduced broods did not respond with reduced feeding rate; nor was parasite prevalence lower among these (Richner et al. 1995). By contrast,

6 363 Reproductive investment and parasite susceptibility feeding rate in Great Tits with reduced broods was found to be reduced compared with a control brood size (Smith et al. 1988; Verhulst 1995). Additionally, König & Schmid-Hempel (1995) experimentally demonstrated that work load in bumble-bee workers was negatively related to immunocompetence. Norris et al. (1994) observed no effect of brood enlargement or reduction on females in terms of parasite prevalence, whereas males tending reduced and enlarged broods were infected more often. They argued that this could be due to a negative covariance between original clutch size and experimentally created brood size, which was not the case in the present study (see Methods). It is not known whether the results from Norris et al. (1994) were related to parental effort, or if individuals were initially a random sample. The present study, however, could not find any statistical evidence for a different response to manipulation between the sexes, but did reveal a lower prevalence of parasites in parents with reduced broods and a higher prevalence of parasites in parents with enlarged broods. The differences between the results from these three studies (Norris et al. 1994; Richner et al. 1995; the present study) may suggest population differences in response to brood size manipulation. First, the three host populations differ in terms of the species of blood parasite most commonly affecting them: Leucocytozoon sp. (Norris et al. 1994), Plasmodium sp. (Richner et al. 1995) and Haemoproteus majoris (Allander & Bennett 1994; this study). Second, there may be cost differences for the host with respect to the defence against different parasite species. Third, in all three cases, experimental studies were performed in only 1 year. Therefore, environmental factors such as food abundance which may differ between years and populations, may have affected the outcome of our respective experiments. However, all three studies are consistent in showing that an increased reproductive effort resulted in a higher prevalence of blood parasites compared with controls. Life-history theory predicts that an investment in current reproduction should be at the expense of future reproduction (Williams 1966). If blood parasites affect the survival probabilities of their hosts (e.g. Richner et al. 1995) or cause a decreased reproductive performance in subsequent breedings (e.g. Gustafsson et al. 1994), they represent a possible mechanism generating the observed patterns of costs of reproduction (reviewed by Lindén & Møller 1989). However, most studies of costs of reproduction in birds reviewed by Lindén & Møller (1989), where brood size have been manipulated, have failed to find any survival and/or fecundity costs. Parents were affected in only 32% of the cases whereas offspring were affected in 60% of the cases. One reason why most studies have failed to find any reproductive costs could be that survival costs may only be evident under severe environmental conditions. Heavily parasitized birds may have a lower survival in bad rather than good years. Furthermore, nothing is known about parasites and disease in the studies reviewed by Lindén & Møller (1989). If parasites are an important factor involved in reproductive trade-offs, one may speculate that bird populations where reproductive costs were evident may be parasitized to a larger extent than populations where no reproductive costs have been found. A trade-off between reproductive effort and parasite defence could mediate trade-offs between reproductive events. An earlier experimental study of the same population of Great Tits as in this study has shown a trade-off between reproductive effort and the probability of laying a second clutch (Lindén 1988) which indicates a reproductive cost. However, blood parasite levels were not measured by Lindén (1988). Birds with experimentally increased brood size had a lower probability of having a second clutch. The fact that both prevalence of parasites and parasite intensity increase with increasing reproductive effort shown in the present study and that Lindén (1988) showed a trade-off between reproductive effort and the probability of laying a second clutch supports the hypothesis that parasites may represent an important portion of the costs of reproduction in this population of Great Tits. Acknowledgements I thank Anders Pape Møller, Ben Sheldon, Anders Berglund, Andrew F. Read, Heinz Richner, Ken Norris, Staffan Ulfstrand and an anonymous referee for valuable comments on the manuscript. I also thank Gordon F. Bennett, who tragically died on 24 December 1995, for analysing all blood smears with excellent skill and speed, Anders Forsman for helping me with the statistics and Dave Wiggins for correcting my English. I am indebted to Michael Gill and Anders Lindholm for assistance in the field. This study was supported by the Royal Swedish Academy of Sciences and the Zoological Foundation. References Alatalo, R.V. & Lundberg, A. (1989) Clutch size in the pied flycatcher Ficedula hypoleuca An experiment. Ornis Fennica 66, Allander, K. & Bennett, G.F. (1994) Prevalence and intensity of haematozoan infection in a population of great tits (Parus major L.) from Gotland Sweden. Journal of Avian Biology 25, Atkinson, C.T. & van Riper III, C. (1991) Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon, and Haemoproteus. Bird Parasite Interactions. Ecology, Evolution and Behaviour (eds J. E. Loye and M. Zuk), pp Oxford University Press, Oxford. 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