1Very few studies to date have evaluated experimentally the effects of blood parasites on physiological variables and breeding performance in wild birds. In this study, blood parasitaemias of female Blue Tits Cyanistes caeruleus L. were experimentally manipulated to assess subsequent changes in immunoglobulin level and parental effort during reproduction.
2At the beginning of the nestling period, female Blue Tits were medicated with a high dose (HD) or a low dose (LD) of the antimalarial Primaquine, or with saline solution (control). Treatment with Primaquine causes a reduction in blood parasitaemias in the study population.
3Immunoglobulin levels decreased in females from the HD group during the experimental period (10 days), while the levels increased in control females.
4Only females in the HD group increased significantly their provisioning rates from the early to the late nestling stage. Total (male and female) provisioning rates increased significantly for the HD and LD groups, but not for the control group.
5Nestlings reared by control females suffered a higher infestation by the ectoparasitic blowfly Protocalliphora azurea (Fallén).
6Medication and the associated decrease in immunoglobulin levels allow females to allocate more resources towards parental effort. In addition, there is a potential link of medication with the health of the nestlings.
7This study gives indirect support to the trade-off between reproductive effort and immune defence in avian hosts, and sheds light on the evolutionary significance of the link between parasitism, immunity, life-history decisions and fitness.
It seems reasonable to predict that costs of parasitism and/or associated physiological responses will show a gradual, fine-tuned adjustment to parasite infection intensity, parasite virulence, health status of individuals or other external environmental pressures. In the present study, we medicated female Blue Tits Cyanistes caeruleus L. with two different doses of Primaquine, an antimalarial chemical compound that has been successfully employed to reduce blood parasitization in this (Merino et al. 2000, 2004; Tomás et al. 2005) and other species (Marzal et al. 2005). A third group of females were maintained as controls. Variation in total immunoglobulin levels was assessed from blood samples. In addition, we measured variation in parental provisioning rates to nestlings and final reproductive performance according to experimental treatment. We predict that female Blue Tit parasitaemia should be reduced according to medication dose, and a similar associated reduction in total levels of circulating immunoglobulins. Our assumption is that concentration in peripheral blood of antibodies reflects the level of general immune defences by hosts (Morales et al. 2004). We also predict that medicated females may be able to expend more provisioning effort as their parasitaemia/immune response is reduced, either because of a reduction of the direct costs imposed by parasites or through a decrease in the costs of maintained immunity. Accordingly, we expect differences among treatments in some measure of nestling health and/or condition.
Materials and methods
The study was carried out during the 2002 breeding season in a Pyrenean Oak Quercus pyrenaica (Willd.) deciduous forest located in Valsaín (Segovia, central Spain, 40°53′N, 4°01′W, 1200 m a.s.l.). A population of Blue Tits breeding in nest-boxes in this area has been studied since 1991 (e.g. Sanz 2002). Every year, nest-boxes are periodically inspected in order to determine reproductive parameters.
Nests matched according to clutch size and laying date (day 1 = April 1) were randomly assigned to one of the following three experimental treatments. Females were trapped in their nest-boxes when their nestlings were 3 days old (hatching date = age 0), and injected subcutaneously with: (1) 0·1 mg of Primaquine (Sigma, St Louis, MO, USA) diluted in 0·1 mL of saline solution (medicated at high dose, hereafter HD); (2) 0·05 mg of Primaquine diluted in 0·1 mL of saline solution (medicated at low dose, hereafter LD); or (3) the same volume of saline solution (controls). Immediately after capture and before injecting, we obtained blood (80–100 µL) from the brachial vein with the aid of a needle and a capillary tube (initial sample). At this capture, female mass was recorded to the nearest 0·05 g with a Pesola (Baar, Switzerland) spring balance and tarsus length was measured with a digital calliper to the nearest 0·01 mm. Females were banded individually with numbered aluminium rings when necessary. To identify the adults on video films (see below), females were ringed with colour rings or painted with red ink on the belly at this capture.
When nestlings were 13 days old, females were recaptured, weighed and blood samples taken (final sample) to determine post-treatment immunoglobulin levels and parasitaemia. Tarsus length of all nestlings was measured with a digital calliper (accuracy 0·01 mm) at 13 days of age and nestling mass was recorded to the nearest 0·05 g. We used mass/tarsus length, as a measure of female and nestling condition. We recorded adult provisioning rates to the offspring by filming nest-boxes for 1 h with a video camera placed several metres away on days 4 and 12 of nestling age. We discarded some observations in which one of the adults was not observed feeding nestlings. Female or total provisioning rates were not related to hour of filming (P > 0·30 in all cases). This was true for both initial and final provisioning rates. After the nestlings had fledged, nests were removed and nest material was carefully dismantled in order to count ectoparasitic Protocalliphora azurea pupae (Merino & Potti 1995a).
blood sample analyses
One drop of blood was smeared on a slide for detection of blood parasites and the remainder of the sample was centrifuged (2000 × g, 5 min) with a portable centrifuge (Labnet, Mini Centrifuge, cat. no. 1201-220V, Woodbridge, NJ, USA). Serum fractions were separated and maintained below 15 °C before being frozen on the same day for later analysis. Blood smears were immediately air-dried and later fixed with ethanol (96%) and stained with Giemsa (1 : 10 v/v) for 45 min. Half of the symmetrical smear was scanned at × 200 magnification in search of large blood parasites such as Trypanosoma, Leucocytozoon or microfilaria, whereas small intraerythrocytic parasites such as Haemoproteus or Lankesterellids (previously reported as Hepatozoon; see Merino et al. 2006) were detected on the other half of the smear using × 1000 magnification (Merino & Potti 1995b; Merino, Potti & Fargallo 1997). Intensity of infection by Haemoproteus parasites was estimated as the number of infected cells per 2000 erythrocytes (Godfrey, Fedynich & Pence 1987). We use presence/absence indexes for Trypanosoma, Leucocytozoon and Lankesterellid parasites due to their low intensities of infection.
Immunoglobulin levels were quantified from plasma samples by direct enzyme-linked immunosorbent assay (ELISA) using a polyclonal rabbit antichicken IgG conjugated with peroxidase (Sigma). Absorbances were measured using a plate spectrophotometer at λ = 405 nm. Details and validation of the method are described in Martínez et al. (2003).
Parametric statistics were used when variables fitted normal distributions. Otherwise, nonparametric tests were employed. Fledging success was analysed by means of a GLZ (Generalized Linear Model; StatSoft 2001) with binomial errors and a logit link. Haemoproteus infection intensities and blowfly pupae number were logarithmically transformed to fit normal distributions. We conducted repeated measures anovas (StatSoft 2001) with treatment as factor to explore changes from initial to final samples in adult variables. We specifically looked for the interaction between treatment and sampling period as a repeated measures factor. Parasite intensity data may be prone to bias even when logarithmically transformed, and thus it has been suggested to assume they adjust to Poisson distributions in some cases (Wilson & Grenfell 1997). However, our data show better adjustments to normal distributions (when logarithmically transformed) than to Poisson distributions. In addition, we have reanalysed the effect of the treatment on final Haemoproteus infection intensity and Protocalliphora abundance by means of GLZ with Poisson errors, and the results do not change (data not shown). As ectoparasites have been shown to affect parental effort (e.g. Hurtrez-Boussès et al. 1998; Merino et al. 1998), we included Protocalliphora abundance as a covariate when analysing provisioning rates. The residuals of the models were tested for normality. All tests are two-tailed. Values presented are mean ± SE.
Two nests (one HD and one control) with a polygynously mated male were discarded from the analysis. Hence, a total of 47 nests were included in the study (17 medicated at LD, 15 medicated at HD and 15 controls). The sample sizes differ between analyses because, in one case the quality of the blood smear did not allow its examination, in three cases the blood sample was insufficient for immunoglobulin analysis and in two cases females escaped before weighing. Both initial and final provisioning rates were recorded for 27 nests (11 from the LD group, seven from the HD group and nine from the control group). However, in some cases we were not able to identify the sex of the adults in one of the two films, and therefore when analysing separately female provisioning rates, sample sizes were reduced to nine nests from the LD group, five from the HD group and four from the control group. Table 1 presents mean (SE) laying and hatching dates, clutch size, female blood parasite infections and immunoglobulin level, nestling and female body condition, female and total (female + male) provisioning rates, and fledging success and number of blowflies for nests in relation to experimental treatment at the beginning and the end of the nestling period.
Table 1. Mean (SE) laying and hatching date, clutch size, female blood parasite infections and immunoglobulin level, nestling and female body condition, female and total (female + male) provisioning rates, fledging success and number of blowflies for blue tit nests in relation to experimental treatment, at the beginning and the end of the reproductive period
HD, females medicated at high dose; LD, females medicated at low dose; control, females injected with saline solution.
Female provisioning rates
Total provisioning rates
Number of blowflies
treatment and infection
Nests assigned to each treatment did not differ with respect to laying or hatching date, clutch size, or brood size at the 3-day nestling stage (P > 0·25 in all cases). At the beginning of the experiment, 89·1% of the females were found to be infected by blood parasites, the most prevalent being Haemoproteus majoris (Laveran) (76·1%), followed by Trypanosoma avium (Danilewsky) (39·1%), Leucocytozoon majoris (Laveran) (30·4%) and a Lankesterellid (21·7%). Only one female appeared infected by microfilaria.
There were no initial differences among treatments in the intensity of infection by Haemoproteus, or prevalences of Leucocytozoon, Trypanosoma or Lankesterellid parasites in females (all P > 0·15). As expected, intensity of infection by Haemoproteus in females decreased differentially among treatments (F2,43 = 4·67, P = 0·015, Fig. 1), in such a way that both HD and LD females experienced a reduction in their parasite load, whereas this was not the case for control females. There were no differences among treatments in final prevalence of infection by Leucocytozoon or Trypanosoma in females (both P > 0·05). However, final Lankesterellid prevalence was significantly lower in HD females as compared with control ones (χ2 = 4·62, P = 0·032).
Before the experiment, females assigned to different treatments did not differ in their immunoglobulin levels (P = 0·111). There was a significant interaction between sampling period and treatment for female immunoglobulin levels (F2,41 = 3·54, P = 0·038, Fig. 2), in such a way that immunoglobulin levels decreased in the course of the experiment in HD females and increased in control females, whereas LD females maintained similar immunoglobulin levels throughout the experiment.
parental condition and provisioning rates
The subsample of nests for which we were able to record provisioning rates did not differ in laying or hatching date, clutch size, or brood size at the beginning of the experiment (P > 0·10 in all cases).
Female condition at first capture did not differ among treatments (P > 0·45). The same was true for initial female and total (male and female) provisioning rates (P > 0·07). The body condition of female Blue Tits decreased from the early to the late nestling stage (F1,42 = 78·10, P < 0·001), but not differentially among treatments (F2,42 = 1·38, P = 0·262). Total provisioning rates increased significantly from the early to the late nestling stage (F1,23 = 6·93, P = 0·015), and differentially among treatments (interaction sampling period × treatment: F2,23 = 4·34, P = 0·025). Total provisioning rates increased significantly from the early to the late nestling stage in the LD and HD groups, but not for the control group. Final total provisioning rates did not differ among treatments (anova: F2,29 = 0·75, P = 0·483).
There was a significant interaction between sampling period and treatment for female provisioning rates (F2,14 = 4·45, P = 0·032, Fig. 3). Females in the HD group increased significantly their provisioning rates from the early to the late nestling stage, while this was not the case for females in the LD and control groups. However, final female provisioning rates did not differ among treatments (anova: F2,21 = 0·39, P = 0·680). Including brood size in the analyses did not change the results for provisioning rates.
effects on nestlings
There were no significant differences among groups in fledging success (Wald statistic = 1·60, P = 0·450) or nestling condition on day 13 (F2,44 = 1·34, P = 0·273). Nevertheless, nests of control females held more blowflies than nests of HD or LD females when controlling for brood size at 13 days of nestling age (treatment: F2,43 = 3·31, P = 0·046, Fig. 4; brood size: F1,43 = 6·76, P = 0·013); the number of blowflies was positively associated with the number of chicks in the nest (r = 0·35; P = 0·016). In addition, the change in intensity of infection (final intensity – initial intensity) by the most prevalent haemoparasite (Haemoproteus) in females, showed a significant negative correlation with nestling condition (r = –0·44, P = 0·002) and a marginally significant positive correlation with the number of blowfly pupae, in the nest (r = 0·25, P = 0·088).
The experimental medication of female Blue Tits with Primaquine was successful in reducing the intensity of infection by Haemoproteus majoris in the HD and LD groups and in reducing the prevalence of infection by Lankesterellids in the HD group, compared with the control group. However, no effect was detected on Leucocytozoon majoris prevalences, in contrast to Merino et al. (2000) and Tomás et al. (2005), possibly because the prevalence of this parasite was lower in the present study. Interestingly, a relatively higher number of birds infected by Lankesterellids in this study may have enabled a reduction in prevalence associated to the medication with Primaquine. Primaquine has been shown to reduce gametocyte density of several species of Plasmodium within 72 h (López-Antuñano 1999; WHO 2001). Apparently, Primaquine acts by binding and modifying the parasite's DNA (López-Antuñano 1999), as well as by disrupting the parasitic mitochondrial membranes (Baird & Rieckmann 2003). As do most antimalarial drugs, primaquine may show dose-dependent, nondesirable side-effects, such as gastrointestinal disturbances and development of methaemoglobinaemia and haemolytic anaemia (Mayorga et al. 1997). However, the mechanism of action of Primaquine is still not fully understood (WHO 2001), and we are not aware of any study reporting direct interactions with the host immune system that may be relevant for the present study. Thus, we can assume that the experimental treatment had the expected effect of a reduction in blood parasitization of female Blue Tits according to Primaquine dose.
We have shown that female immunoglobulin levels changed differentially across the experiment depending on the treatment females received. We assume that the observed change in immunoglobulin level is associated with the experimental reduction in haemoparasite load as a positive relationship between immunoglobulin levels and haemoparasite infection as has been previously reported (Isobe & Suzuki 1987; Ots & Hõrak 1998; Morales et al. 2004; Tomás et al. 2005). Thus, control females needed to increase their levels of circulating immunoglobulins during the nestling period, while females in the LD group showed similar levels and females in the HD group could reduce their immunoglobulin levels. It is possible that control females needed to increase their antibody levels to manage the infection and buffer its detrimental effects. In contrast, HD females, through being released from the drain imposed by parasites, could reduce the resources devoted to the immune function (as measured by circulating immunoglobulin levels), an important saving given the resources required for eliciting and maintaining an immune response (Ilmonen et al. 2000; Lochmiller & Deerenberg 2000; Moret & Schmid-Hempel 2000; Råberg et al. 2000; Bonneaud et al. 2003; Hanssen et al. 2004; see also Verhulst, Riedstra & Wiersma 2005). This trade-off could be particularly important in periods of more intense energetic effort such as the nestling period in altricial birds. Although final immunoglobulin levels did not differ among groups, we have detected an effect of the treatment by exploring the changes between initial and final samples within groups. This indicates that individuals may not have the same basal level of immunoglobulins. The cost associated with the experiment is thus detected as a change in immunoglobulin levels in the course of the nestling period, so the observed change in provisioning rates is more likely associated with this change, rather than with variation in final levels. This indicates the importance of monitoring initial health state of birds in detecting reproductive trade-offs (Sanz et al. 2002).
The reduction in parasite load and circulating levels of immunoglobulins has probably allowed females of the HD group to increase their parental effort in the course of the nestling period, something that females in the LD and control groups could not afford (Merino et al. 2000). Although final provisioning rates did not differ among groups, we have shown that control birds are unable to increase their provisioning effort from the early to the late nestling stages. It should be noted that there were no differences among treatments in female condition. The reduction in effort in individuals with higher parasitaemias might be adaptive if it allows them to overcome infection and to increase survival prospects (Bonneaud et al. 2003). It is known that reduced activity might conserve energy and permit tissue repair during infection (Hart 1988). This result could be consistent with the existence of a trade-off between energy allocated to reproductive effort and to parasite control/immune investment (Sheldon & Verhulst 1996).
We did not find any direct detrimental effect of female parasitization on fledging success or nestling condition in relation to the experimental treatment. However, there is a link between female haemoparasite load and health state of nestlings as shown by nest infestation by Protocalliphora azurea larvae. Nests attended by control females contained more blowflies than nests of HD or LD females. The difference observed among treatments is presumably mediated by parental effort causing differences in nestling susceptibility to infestation with ectoparasites (Hudson & Dobson 1997) or by differences in time devoted by the parents to nest sanitation activities (Hurtrez-Boussès et al. 2000). Similarly, maternal effort has been reported as mediating the prevalence of trypanosomes in the offspring of another hole-nesting passerine (Merino, Potti & Moreno 1996). This is probably because poorly nourished nestlings are less able to mount an efficient immune response (e.g. Merino et al. 1996; Hudson & Dobson 1997; Saino, Calza & Møller 1997; Saino et al. 2002b), and the same rationale may be applied to our results: an increase in feeding effort by females may allow nestlings to attain better defences. Indeed, that the change in intensity of infection by the most prevalent haemoparasite (Haemoproteus) in females showed a significant negative correlation with nestling condition and a marginally, significant positive correlation with the number of blowfly pupae in the nest, adds support to the idea that female parent parasitaemias have detrimental effects on the offspring, which is in accordance with previous findings (Merino et al. 2000; Tomás et al. 2005).
To conclude, we have demonstrated for the first time that experimental reduction in blood parasitaemias allow avian hosts to save resources devoted to immune function and invest those resources in reproductive activities. In addition, there is a potential link of medication with the health state of nestlings. This study gives indirect support to the trade-off between reproductive effort and immune defence in hosts, and sheds light on the evolutionary significance of the link between parasitism, immunity, life-history decisions and fitness (Norris & Evans 2000). The combined experimental manipulation of both haemoparasite load and immune response (the latter, ideally by challenging birds with isolates of the same, dead parasite) in a wild avian population might help to separate the costs associated with the damage caused by the parasite to that of eliciting an immune response against it in an ecological framework.
We thank Javier Donés (Director of ‘Montes de Valsaín’) for permission to work in the study area. The Junta de Castilla y León authorized the ringing and handling of birds. This study was funded by projects BOS2000-1125 and BOS2003-05724 from Ministerio de Ciencia y Tecnología (to SM) and BOS2001-0587 and CGL2004-00787 (to J. Moreno). We thank Tonantzin Calvo, László Z. Garamszegi, José Llama, Javier Martínez and Inma Nogueras for their help. J. Morales was supported by a FPI grant from Ministerio de Ciencia y Tecnología. GT was supported by a FPI grant from the Comunidad de Madrid and partially by an accommodation grant to reside at the Residencia de Estudiantes from the Ayuntamiento de Madrid and an I3P post-doctoral contract from CSIC. This study is a contribution to the research developed at the El Ventorrillo field station. We thank two anonymous reviewers and the Associate Editor for constructive comments on the manuscript, and L.M. Carrascal for kindly providing statistical advice.