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Keywords:

  • Haemoproteus;
  • heat-shock proteins;
  • parasites;
  • parental investment;
  • reproductive stress

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Physiological stress in animals may impose a limit for investment in current reproduction in the wild. A brood manipulation experiment was conducted in a population of blue tits Cyanistes caeruleus to study the effect of parental effort on changes in two types of proteins related with stress: the blood stress protein HSP60 and the plasma immunoglobulins.
  • 2
    Levels of HSP60 were reduced across the experiment for females attending reduced broods, and females attending enlarged broods experienced a reduction of immunoglobulin levels. Moreover, the overall changes in the levels of both proteins were positively related.
  • 3
    By controlling for the change in immunoglobulin levels we found an increase in HSP60 for females in the enlarged treatment, presumably to offset deleterious effects derived from increased effort.
  • 4
    Maternal effort was able to partially compensate for the effect of treatment as nestlings did not differ in mass and levels of immunoglobulins and HSP60 among treatments.
  • 5
    Physiological stress as reflected in stress and immunoglobulin proteins may limit maternal effort in breeding blue tits.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In temperate areas small passerines are short-lived and therefore have a limited number of opportunities to reproduce. Therefore they may be selected to maximize their investment in current reproduction without jeopardizing survival before juvenile independence (Tuomi 1990; Moreno 1993). If parental effort is modulated to avoid high survival costs for parents during current reproduction, one would expect that birds were working close to their own limit of effort, different for each individual during each breeding season (Peterson, Nagy & Diamond 1990; Potti, Moreno & Merino 1999). The consequence of experimental alteration of brood sizes in small passerines is that, although more young are usually fledged from enlarged broods, offspring fare less well (Gustafsson & Sutherland 1988; Lindén & Møller 1989; Smith, Källander, & Nilsson 1989; Tinbergen & Boerlijst 1990). On the other hand, parents attending reduced broods lower their investment following brood reduction (Forbes 1993; Ploger 1997). Although many studies confirm this pattern (Stearns 1992) the mechanisms involved are far from clear. In this sense, the frequently observed parental reluctance or inability to increase their work level may be related to the risks for immediate survival involved in surpassing naturally high levels of physiological stress.

Parental effort has been shown to impose high costs (Lindén & Møller 1989; Roff 1992; Moreno 1993, 2002) and intense physical effort induces the production of metabolites, such as oxygen-free radicals, capable of damaging cell function (Alonso-Alvarez et al. 2004; Wiersma et al. 2004). A series of responses to these metabolites are in consequence triggered in organisms. Among the more basic and evolutionary conserved responses to maintain cellular homeostasis is the production of so-called heat-shock proteins (HSPs). These intracellular proteins, now more generally termed stress proteins, play an important part in the correct folding of newly synthesized proteins needed during responses to many external stressors and therefore, their role is to maintain cell function during stressful periods (Morimoto 1991). These stress proteins are able to respond to a variety of stressors ranging from pollutants to parasites (see review in Sørensen, Kristensen & Loeschcke 2003) and can also increase in response to metabolic demands by cells (Welch 1992; Merino et al. 2004). The ecological significance of these proteins is still not well-known, but their important function and their presence in all living beings so far studied indicate that they may play a major role in buffering stress in the wild.

Parasitic disease may also influence the capacity of adult birds to invest in current reproduction in several ways (Møller 1997; Merino et al. 2000). There exists a clear relationship between parasitism and immune and stress responses. Several studies have shown that birds suffering the effects of parasites show higher level of stress proteins (Merino et al. 1998a, 2002; Tomás et al. 2005), although the ultimate cause of the cellular response is not known. On the other hand, immune responses against parasites have been thoroughly studied (Wakelin & Apanius 1997) and increased levels of immunoglobulins have been reported in response to haemoparasite infections (Isobe & Suzuki 1987; Ots & Hõrak 1998; Morales et al. 2004). In addition, intense exercise may induce immunosuppression (Råberg et al. 1998; Pedersen & Hoffman-Goetz 2000) and individuals may be therefore more susceptible to diseases. Moreover, birds are known to suffer from relapses by several diseases during reproduction as is the case with malaria-like infections (Atkinson & Van Riper 1991). Immune defences are also affected by reproductive effort (Hõrak, Ots & Murumägi 1998; Moreno, Sanz & Arriero 1999b; Hasselquist, Wasson & Winkler 2001; Saino et al. 2002; Apanius & Nisbet 2006), and this variation may affect antiparasite resistance (Nordling et al. 1998) and even survival (Hanssen et al. 2004).

We here study the variation in the levels of intracellular HSP60 and serum immunoglobulins in parental peripheral blood as two different indirect indicators of reproductive costs, following an experimental manipulation of brood size in a small short-lived passerine, the blue tit Cyanistes caeruleus (Linnaeus 1758). We focus on females because, contrary to males, they are sure of their maternal investment in current reproduction and we can expect higher responses to manipulation of brood size. We expect an immunosuppressive effect of increased parental effort that can be reflected in a reduction in immunoglobulin levels as shown for specific immune responses in previous studies (Deerenberg et al. 1997; Nordling et al. 1998), and an increase in the stress protein HSP60 in response to higher metabolic demands and their associated costs for cells (Wiersma et al. 2004). In addition, immune system and stress proteins interact (Kaufmann 1990; Haas 1994), and both types of proteins may also compete for resources for their production. Moreover, during stress, when host heat-shock proteins are expressed at higher levels, the risk of autoimmunity increase due to the similarity among pathogen and host HSPs and therefore it is expected that the immune system was suppressed to avoid risks of immunopathology (Råberg et al. 1998; Svensson et al. 1998). Therefore, we explore the potential effect of each type of protein on the other. Physiological responses by nestlings will differ among treatments in the case that parental effort cannot buffer the effect of brood size manipulation.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

During the spring of 2003 we conducted an experiment in a population of blue tits breeding in nest boxes in Valsaín (Segovia), Central Spain (40°53′N, 4°01′W). Reproduction was monitored by periodical inspection of nests. Three days after hatching female birds identified by the presence of the brood patch were captured attending nests, and blood was sampled from the brachial vein (80–100 µL). Birds were ringed with aluminium rings for individual recognition and weighed to the nearest 0·1 g with a spring balance. During the same day, two nestlings were transferred from one nest (hereafter reduced nests) to another (hereafter enlarged nests), while a third nest of the same hatching date was maintained as control. Each triad of nests had a similar (± 1 nestling) original number of nestlings. Ten days later females were recaptured, sampled for blood and reweighed. Nestlings at the age of 13 days (hatching day = day 0) were weighed and sampled for blood as described for adult females.

blood sample analyses

One drop of each blood sample from adult females was immediately smeared on a microscopy slide and air-dried. Smears were fixed with 95% ethanol and stained for 40 min with Giemsa stain (1/10 v/v in phosphate buffer). Slides were scanned for presence of blood parasites as described by Merino, Potti & Fargallo (1997) and Haemoproteus quantified as the number of infected cells per 2000 erythrocytes (Godfrey, Fedynich & Pence 1987). Other blood parasites are present in low densities and or prevalences and therefore not included in analyses (see Tomás et al. 2005).

The rest of the blood sample was centrifuged and plasma and blood cells were separated and frozen for later analyses. Plasma samples were used to quantify immunoglobulin (IgG = IgY) levels by a direct enzyme-linked immunosorbent assay (ELISA) using a polyclonal rabbit antichicken IgG conjugated with peroxidase (Sigma, St Louis, MO, USA). Absorbances were measured using a plate spectrophotometer at λ = 405 nm. Details and validation of the method are described by Martínez et al. (2003). Average immunoglobulin levels of nestlings in a brood were obtained by mixing the same volume of serum from each nestling from a nest in order to minimize the number of analyses.

The cellular fraction of blood was used to quantify the stress protein HSP60 by means of Western blotting using monoclonal antibodies anti-HSP60 (clone LK2, Sigma) as primary antibodies and a peroxidase-conjugated secondary antibody (Sigma) as described in Merino et al. (1998a) and Tomás, Martínez & Merino (2004). Protein bands were quantified using image analysis software for windows (Scion Corporation, Frederick, Margland, USA). Immunoreactivities (arbitrary units) were obtained as: immunoreactivity = area × mean intensity of the band. Average levels of HSP60 for nestlings of the same brood were obtained by mixing the same quantity of total blood protein of each nestling from a nest.

statistics

In order to check for changes between initial and final blood stress protein and immunoglobulin levels within individual females from each treatment we conducted repeated measures ancovas with treatment (brood manipulation) as factor. We specifically looked for the interaction between treatment and the sampling period (repeated measures factor). Previous experiments and correlative studies have demonstrated that Haemoproteus intensity and prevalence is related with HSP60 and IgG levels (Morales et al. 2004; Tomás et al. 2005). Therefore we control for the potential effect of infection on protein changes by introducing as a covariate the average intensity of infection by Haemoproteus, calculated as the sum of initial and final intensities divided by two. As intensity of infection did not fulfil requirements of parametric tests, data were log-transformed. We also checked for the effect of the change of each type of protein on the other by introducing final less initial values of one of them as a covariate in the repeated measures analyses of the other. Means are presented with SE. Analyses were performed with Statistica 6·0 (StatSoft, Inc. 1984–2001, Tulsa, OK, USA). As variation in initial clutch size is controlled by experimental design we do not include this factor in the analyses. Controlling by initial clutch size did not alter conclusions.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Overall, 29 triads of nests were included in the experiment. The maximum sample size available was used for each analysis although sample sizes vary because three females from each treatment were not recaptured and the initial plasma sample of one female was insufficient for immunoglobulin analyses. In addition, nestlings from three nests died before 13 days of age and we did not obtain blood from nestlings of another nest. Therefore sample sizes are 87 initial and 78 final samples for HSPs and 86 initial and 78 final samples for IgGs. HSPs and IgGs were analysed from nestlings of 83 nests.

Nests assigned to each treatment did not differ with respect to clutch size, hatching date and number of nestlings prior to the brood manipulation (F2,84 = 0·45, P = 0·64, F2,84 = 0·01, P = 0·99 and F2,84 = 0·24, P = 0·78, respectively). At the end of the experiment, brood sizes differed among experimental treatments as expected (reduced broods, 6·04 ± 0·30, control broods, 7·39 ± 0·35, enlarged broods, 9·03 ± 0·50, F2,82 = 14·39, P < 0·0001).

The more prevalent and abundant blood parasite in the population was Haemoproteus majoris, infecting 73·6% of female blue tits. Intensity of infection in females averaged 18·52 ± 2·59 infected cells per 2000 erythrocytes for initial samples and 14·65 ± 2·34 per 2000 erythrocytes for final samples. Intensity of infection decreased across the experiment (F1,74 = 6·17, P = 0·02), but not differentially for each experimental group (F2,74 = 1·20, P = 0·31). Initial or final levels of infection did not differ among experimental groups (F2,84 = 0·017, P = 0·98 and F2,74 = 0·14, P = 0·87, respectively).

There existed a significant interaction between the sampling period and treatment for HSP60 [F2,75 = 3·21, P = 0·046 (Fig. 1a), Fisher LSD Post-Hoc test show that initial levels of HSP60 from the three experimental groups differ significantly only from final levels of HSP60 of the reduced group, P < 0·02], showing that a significantly greater reduction in levels of HSP60 occurred between the initial and final samples for females attending reduced broods as compared with females attending control and enlarged broods. Introducing average intensity of infection by Haemoproteus as covariate did not change the result (treatment × sampling period: F2,73 = 4·09, P = 0·021; infection × sampling period: F1,73 = 1·38, P = 0·24). Average initial levels of HSP60 in females did not differ (F2,84 = 0·16, P = 0·85), but a tendency towards a lower level for females in the reduced group appeared for final samples (F2,73 = 2·76, P = 0·07).

image

Figure 1. (a) Changes in HSP60 levels for female blue tits according to experimental treatment. Bars are SE. (b) Changes in immunoglobulin levels for female blue tits according to experimental treatment. Bars are SE.

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For immunoglobulins, the interaction between treatment and the sampling period was also significant (F2,74 = 3·45, P = 0·037, Fig. 1b, Fisher LSD Post-Hoc test show that only initial IgG for enlarged group differs from final IgG of the same group, P = 0·038), showing a reduction in immunoglobulins throughout the experiment for females attending enlarged broods as compared with females in the two other treatments. Controlling for Haemoproteus infection did not affect this conclusion (treatment × sampling period: F2,72 = 3·34, P = 0·041; infection × sampling period: F1,72 = 1·74, P = 0·19). However, average initial or final levels of immunoglobulins in females did not differ among experimental groups (F2,82 = 0·17, P = 0·84 and F2,73 = 0·94, P = 0·40, respectively).

The relationship between initial levels of both immunoglobulins and HSP60 proteins was significant (F1,82 = 4·04, P = 0·048; treatment: F2,82 = 0·34, P = 0·71), but not for final levels of both proteins (F1,74 = 0·08, P = 0·77; treatment: F2,74 = 2·11, P = 0·13). However, the magnitude of the change (final less initial level) of each type of protein was directly related to the magnitude of change of the other (F1,73 = 14·00, P < 0·001; Fig. 2). Also, treatment showed a significant effect, with the magnitude of the change for HSP60 increasing from females attending reduced to females attending enlarged broods (F2,73 = 5·91, P = 0·004) and immunoglobulin levels being lower for enlarged broods as compared with both controls and reduced broods (F2,73 = 6·21, P = 0·003). Similar results were obtained by introducing the change between initial and final levels of immunoglobulins as a covariate in the repeated measures analyses: we found a reduction in HSP60 for control and reduced groups and an increase for the enlarged ones (F2,73 = 5·91, P = 0·004). Conversely, by introducing the change between initial and final level of HSP60 as a covariate in the analysis of immunoglobulins we found an increase for the control and reduced groups and a marked decrease for enlarged ones (F2,73 = 6·22, P = 0·003). Clutch size did not affect significantly to initial or final levels of both proteins (P > 0·19 in all cases, data not show).

image

Figure 2. Relationship between change (final less initial) in immunoglobulin and HSP60 levels.

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Female mass decreased across the experiment (F1,70= 154·42, P < 0·0001), but differences among treatments were not detected (F2,70 = 0·19, P = 0·82). Average nestling mass at the end of the experiment showed a tendency to differ between experimental treatments (F2,81 = 2·49, P = 0·09), implying that nestlings from reduced broods tended to be heavier than those of control and enlarged broods. Average immunoglobulin and HSP60 levels of nestlings did not differ between treatments (F2,80 = 0·53, P = 0·59 and F2,80 = 0·34, P = 0·71, respectively) and were not significantly correlated (r = 0·001, P = 0·99).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Several studies have investigated the relationship between hormonal measures of stress and parental effort in birds. Saino et al. (2002) showed that male barn swallows with large sexual ornaments had relatively low corticosterone levels at the end of breeding period, suggesting that those males were less exposed or susceptible to stress due to parental effort. On the other hand, Ilmonen et al. (2003) found that corticosterone tends to increase with high parental work load in male pied flycatchers, indicating that parental effort alters stress levels and the physiological state of birds. The experiment presented here reports the first evidence that parental effort affects general physiological stress in birds as measured with stress proteins. Females attending reduced broods showed a significant decrease in the level of blood stress protein HSP60 across the experiment, while females from control and enlarged nests did not change significantly their levels of HSP60. This difference may imply that females are naturally working close to their maximum level of cellular stress, so that only a decrease in their work load allows them to reduce the cellular stress response. Based on the known function of stress protein HSP60 in other organisms, responding to different stresses at the level of mitochondria (Welch 1992; Hartman & Gething 1996), we can speculate that the reduction in the number of nestlings being raised produces a reduction in the metabolic activity of the individual female (Deerenberg et al. 1995) that is finally expressed in the activity of blood cell mitochondria, where HSP60 is implicated in the correct folding of newly synthesized proteins (Voellmy 1996). The reduction in stress proteins is maintained while controlling for the potential effect of infection, indicating that the consequences of brood manipulation are more important than those of infection for the production of stress proteins. However, the final level of HSP60 did not differ significantly between groups, thus pointing out that the limit for reduction in the levels of this protein was narrow, probably because lower levels of proteins were inadequate to maintain cellular homeostasis in the presence of several stressors acting during reproduction. The need of a certain level of HSP60 for controlling the effects of infection may prevent attaining lower levels of this protein in cells (see Tomás et al. 2005); however, an increase in HSP60 response may be limited by the costs of over-expression of this protein (Merino et al. 1998a; Råberg et al. 1998; Sørensen et al. 2003). In any case, control females are apparently working at stressful levels during nestling care.

The different function, mode of action and ways of regulation of immunoglobulins with respect to the stress protein may well explain why a reduction in immunoglobulins occurred for birds attending enlarged broods but in HSP60 for birds attending reduced broods. Immunoglobulins are down-regulated by extra effort in a complex interaction with hormones released during exercise and reproduction (Pedersen & Hoffman-Goetz 2000). However, stress proteins are intracellular proteins involved in response to many different kinds of stressors, and their expression will be expected to increase in response to extra effort in order to buffer effects of oxidative stress and infection following a reduction in immunoglobulins. The production of one type of protein may be limited by the production of the other if they are competing for nutrients or energy available or if the synthesis of one of them depends on the production of the other, as is the case for some stress proteins implicated in the assembly of immunoglobulins (Kaufmann 1990; Haas 1994). The marginally significant relationship between initial levels of both types of proteins and the positive association between the magnitudes of their changes may imply that HSP60 is in some way involved in the folding or activation of immunoglobulins. This is the first evidence of such an association in a natural population of vertebrates. However, the lack of a significant relationship for final levels of both proteins indicates that the treatment altered that relationship, probably because of the role of HSP60 in other functions maintaining cell homeostasis (Welch 1992). Therefore, when the association between changes in HSP60 and in immunoglobulins was controlled for, a greater increase in HSP60 in response to brood enlargement than in the other treatments was detected. The increase in HSP60 is associated to a reduction of immunoglobulin levels giving some support to the hypothesis of immunosuppression mediated by energy requirements (Sheldon & Verhulst 1996), although the fact that HSP60 may provoke autoimmune responses (Yang & Feige 1992) may imply that reduction in immunoglobulins is linked with immunopathology avoidance (Råberg et al. 1998) and not to a competitive inhibition between proteins.

Several studies have shown that immunoglobulin levels reflect susceptibility to stress and parasite infection (Ots & Hõrak 1998; Morales et al. 2004) as well as survival prospects (Christe et al. 2001) and reproductive performance (Apanius & Nisbet 2006). Higher levels of IgG can be regarded as a greater allocation of host resources to this immune mechanism (Apanius & Nisbet 2006). In addition, reproductive effort has been shown to negatively affect antibody responsiveness to specific antigens (Deerenberg et al. 1997; Nordling et al. 1998). This may be related to depressed antibody production generally, as suggested by our result concerning total immunoglobulins in blood for females attending brood-enlarged nests.

As shown by previous studies (Nur 1984; Deerenberg, de Kogel & Overkamp 1996) we assume that birds attending enlarged broods increased their parental effort with respect to those attending control and reduced nests. Although no effect of manipulation was detected in female mass changes, parental reproductive costs may be detected in terms of physiology (Hõrak et al. 1998; Wiersma et al. 2004; this study) or of survival (Nur 1984; Hõrak 1995). Enlarged broods produced more nestlings than control ones but with similar immunoglobulin and HSP60 levels. Therefore, presumable increases in parental effort are apparently able to compensate for increments in brood demands with respect to nestling condition.

The ability to buffer stress by means of physiological responses may allow individuals to cope with high energy demands and effort during reproduction. Short-lived small passerines have a limited number of opportunities for reproduction and therefore should maximize their effort in each reproductive event, but without putting immediate survival at risk (Tuomi 1990; Moreno 1993). Accordingly, several studies have shown constraints on current reproductive effort in small passerines (Alatalo, Lundberg & Stahlbrandt 1982; Nur 1984; Smith et al. 1988; Eguchi 1993; Moreno et al. 1995, 1999a; Merino et al. 1998b), pointing out that parents are normally working near to their maximum level of exertion. By using a measure related to response to stress in blood, we show that birds being forced to rear two extra chicks are unable or reluctant to increase their levels of stress defences, and when this is accomplished, it is by a reduction in immunoglobulins, thus compromising immune function. Any reduction in brood demand is immediately translated into reduced stress protein synthesis probably due to the fact that production and maintenance of proteins in response to stress are costly (Merino et al. 1998a; Svensson et al. 1998; Sørensen et al. 2003; Martínez, Merino & Rodríguez-Caabeiro 2004), and therefore birds should reduce their levels whenever possible. Physiological responses to stress and diseases may therefore impose a limit to reproductive investment in blue tits. The experimental alteration of levels of stress proteins may help to disentangle the mechanisms that relate stress protein and immunoglobulin levels.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was financially supported by projects BOS2003-05724 to S. Merino and J. Martínez and CGL2004-00787/BOS to J. Moreno (Ministerio de Ciencia y Tecnología). J. Morales and J. Martínez de la Puente were supported by a FPI grant from MCYT and ‘El Ventorrillo’ Field Station grant from CSIC, respectively. G. Tomás is supported by an I3P contract from CSIC. We also thank Tonantzin Calvo and Consuelo Corral for help in the field. We were authorized by Javier Donés, Director of Centro Montes de Valsaín to work in the study area. Dirección General del Medio Natural (Junta de Castilla y León) authorized the capture and ringing of birds. This paper is a contribution from the ‘El Ventorrillo’ field station. Comments and suggestions by Victor Apanius and an anonymous reviewer considerably improve a previous version of the manuscript.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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