Habitat Effects on Physiological Stress Response in Nestling Blue Tits

195

Habitat Effects on Physiological Stress Response in Nestling Blue Tits Are Mediated through Parasitism Elena Arriero1,* Juan Moreno1 Santiago Merino1 Javier Martıńez1,2 1 Departamento de Ecologıá Evolutiva, Museo Nacional de Ciencias Naturales–Consejo Superior de Investigaciones Cientı´ficas, Jose´ Gutie´rrez Abascal 2, E-28006 Madrid, Spain; 2 Departamento de Microbiologıá y Parasitologıá, Facultad de Farmacia, Universidad de Alcala´, E-28871 Alcala´ de Henares, Spain Accepted 7/25/2007; Electronically Published 1/15/2008

ABSTRACT We investigated determinants of the physiological stress response mediated by stress proteins in blue tit Cyanistes caeruleus nestlings growing up in oak forests in central Spain, resulting from different forest management practices. We assessed circulating levels of the heat-shock protein HSP60 as an integrated physiological measure of the conditions experienced by nestlings during postnatal development. The effects of habitat quality and parasite infections on nestling rearing environment were then assessed through this measurement of stress response. Our results showed that newly acquired ecto- and hemoparasite infections were associated with forest habitat structural characteristics, higher prevalence of fleas and blood parasites in more mature forests, and higher prevalence of blowflies in degraded forests. While habitat characteristics did not explain variation in stress protein levels, infestation by blowfly larvae of the genus Protocalliphora and hematozoa infection by Leucocytozoon were significantly associated with higher levels of HSP60. Thus, upregulation of the expression of certain stress proteins seems to be a common physiological mechanism to alleviate the negative impact of parasite infections in growing birds. Habitat characteristics may thus indirectly determine growth conditions for forest birds mediated through their association with one of the most important selection pressures for offspring development, parasite infections. * Corresponding author. Present address: Unite´ Mixte de Recherche 7103, Laboratoire de Parasitologie Evolutive, Universite´ Pierre et Marie Curie, Paris 6, F-75005 Paris, France; e-mail: [email protected]. Physiological and Biochemical Zoology 81(2):195–203. 2008. 䉷 2008 by The University of Chicago. All rights reserved. 1522-2152/2008/8102-07014$15.00 DOI: 10.1086/524393

Introduction Developmental period is one of the most vulnerable stages in the life cycle of an organism, and the environmental conditions experienced early in life can have important fitness consequences (reviewed in Lindstro¨m 1999; Metcalfe and Monaghan 2001). Environmental conditions determine extrinsic and intrinsic selection pressures that constrain growth (reviewed in Starck and Ricklefs 1998). Altricial birds, in particular, experience a posthatching period of development confined to the nest and completely dependent on the parental care provided by their progenitors. Thus, nestlings can exhibit considerable variation in developmental patterns due to differences in rearing environment (Ardia 2006). Environmental factors may impact nestling growth and survival at different scales. Short-term sources of variation, such as transient severe weather conditions, can cause rapid variation in food supplies or reallocation of body resources to homeothermy (reviewed in Starck and Ricklefs 1998). This type of variation may affect mass gain and growth, though stored reserves can normally compensate for these temporary harsh circumstances. On the other hand, environmental factors that persist during the whole developmental period may have severe impacts on nestling growth and survival or impair future reproductive performance (Lindstro¨m 1999; Romero 2004). Among these sources of chronic stress, parasitic infections are known to cause major problems for breeding birds and their offspring (reviewed in Møller 1997). Thus, habitat quality, through its biotic and abiotic components, can be an important source of variation in nestling growth acting on a long-term basis (Tremblay et al. 2003). In this context, habitat structural features can be used as an integrated measure of environmental conditions for growing forest birds. Critical aspects such as food availability, predation risk, and microclimatic conditions may be a function of vegetation structure and conservation state of the forest (reviewed in Brokaw and Lent 1999). Given the wide variety of sources of variation in rearing conditions for nestlings, a broad spectrum of adaptations to reduce the impact of poor rearing conditions on nestling fitness is expected. Physiological adaptations determine the range of environmental conditions under which individuals can persist without jeopardizing fitness (Wikelski and Cooke 2006). The physiological reactions that organisms have evolved to minimize the detrimental effects of stressful environmental conditions are known as the stress response (reviewed in Buchanan

196 E. Arriero, J. Moreno, S. Merino, and J. Martıńez 2000). The ability to mount such responses and their level of activation are generally associated with overall health (Romero 2004). Thus, the effects of habitat quality and other environmental factors on the general physiological state of an organism may be assessed through individual variation in physiological mechanisms of the stress response. In this context, physiological indicators of stress may be used as an integrated assessment of the conditions experienced by nestlings during postnatal development (Suorsa et al. 2003). Individual levels of stress can be assessed in a wide variety of ways. Heat-shock proteins (HSPs) represent a reliable method for assessing stress in free-living animals and are increasingly used in avian studies (Eeva et al. 2000; Moreno et al. 2002; Garamszegi et al. 2006; Morales et al. 2006). Circulating levels of HSPs have shown to be correlated with other indicators of stress, such as the widely used heterophil/lymphocyte ratio (Moreno et al. 2002). HSPs, or stress proteins, are highly structurally conserved proteins produced by cells in response to a wide range of potential stressors (Buchanan 2000). Their function as molecular chaperones plays an important role in maintaining cellular homeostasis and preventing protein misfolding during stress challenges (Feder and Hofmann 1999). However, overexpression of HSPs is known to have deleterious consequences (Feder and Hofmann 1999). The first stressor reported was heat, but an increasing number of stressful agents, such as disease, heavy metals, parasites, and inbreeding, have been reported to induce the expression of these proteins in a wide variety of taxa (reviewed in Sørensen et al. 2003). In wild birds, high levels of HSPs have been associated with parasite infections (Merino et al.1998), poor nestling growth (Moreno et al. 2002), sibling competition (Martıńez-Padilla et al. 2004), and reduced immunocompetence (Morales et al. 2006). In addition, experimental manipulation of ectoparasite or hemoparasite loads in birds has been associated with differences in HSP levels between experimental and control individuals (Merino et al. 1998; Toma´s et al. 2005). Thus, the expression of this type of protein in response to chronic and less severe sources of stress is ecologically relevant and can be an important physiological mechanism for coping with stressful conditions (Feder and Hofmann 1999; Sørensen et al. 2003). The blue tit (Cyanistes caeruleus) is a small (11 g) holenesting passerine of European woodlands, exceptionally successful at exploiting a wide range of habitats (Cramp and Perrins 1993). During the breeding season (typically mid-April to mid-June), adults defend territories (0.16–0.81 ha), but they are gregarious year-round. Females build the nest and incubate alone, although both parents feed the young (Cramp and Perrins 1993). In the study population, laying date begins in late April, clutch size ranges from two to 14 eggs, with an average of nine, and the average number of fledglings per pair is eight (Arriero et al. 2006). This study aims to explore the role of HSPs as indicators of sources of chronic stress during development in nestling blue tits. We aim to test to what extent variation in the physical environment (i.e., habitat structure, impact from parasites) can

be reflected in nestling physiology. Sex-biased differences in the effects of parasitism on nestlings have been reported for other passerine species (Potti and Merino 1996; Badyaev et al. 2006). Thus, there might be sexual differences in the physiological mechanism of stress response to parasitic infections. In particular, we predict that (1) circulating levels of HSP60 shortly before fledging will be associated with variation in habitat characteristics; (2) higher levels of stress proteins will be associated with parasitic infections; and (3) other factors, such as hatching date and brood size, will predict variation in stress protein levels through their association with food availability. Methods The study was conducted during the breeding seasons of 2001 and 2002 in deciduous forests of Pyrenean oak Quercus pyrenaica in central Spain (40⬚49⬘N, 3⬚56⬘E). Pyrenean oak forests are the characteristic deciduous woodlands of montane areas in central Spain (Costa et al. 1998) and the preferred breeding habitat for the blue tit in this region (Dıáz 2003).

Habitat Characteristics To examine the role of forest features influencing rearing conditions for the offspring, we placed a total of 600 nest boxes in Pyrenean oak forest plots differing enormously in vegetation structure. Young, degraded, and open-forest plots resulting from intense cattle grazing were compared with mature, wellpreserved, and structurally more complex forest plots. Forest plots were defined as areas with spatial homogeneity in vegetation structure. Three 20-ha forest plots from each category (young or mature forests) were located in the area, and 100 nest boxes were placed in each of the six patches. Wooden nest boxes (12 cm # 12 cm # 20 cm, 3-cm diameter entrance hole) were placed resting on tree branches at approximately 3 m above ground level in a grid of 30–50 m between adjacent boxes. Structural features of the forest were measured in a total of 284 points (50-m diameter) throughout the six forest plots. All points were located and centered in occupied nest boxes, thus sampling vegetation characteristics of breeding territories. The structural variables included estimates of vertical profiles (shrub and tree layer height) as well as horizontal profiles (density of juvenile trees !10 cm diameter at breast height [dbh], density of old oak trees 130 cm dbh, and average diameter of the five trunks with the highest dbh). These variables were the ones that best showed the variation in habitat structure at the geographical scale selected in our study. A principal components analysis was used to extract a single factor of forest structural complexity. The factor explained 50.5% of the variation in habitat structure (eigenvalue p 2.53 ) and defined a gradient of forest structural complexity. Lower values of the factor represented successionally young and heavily managed areas (reduced density of thick trunks, lower average tree height, and smaller trunk diameter). Factor loadings were as follows: shrub height p 0.293, tree height p 0.784, trunks 5–10

Habitat, Parasites, and Stress Proteins in Growing Birds 197 cm p ⫺0.08, trunks 130 cm p 0.812, and average trunk diameter p 0.898. The two categories of forests considered in this study differed significantly in forest structural complexity factor (F1, 283 p 259.06, P ! 0.001; young: ⫺0.702 Ⳳ 0.04, N p 140; mature: 0.678 Ⳳ 0.07, N p 145). Arriero et al. (2006) provides a thorough description of the vegetation measurements. All study sites were located between 1,000 and 1,500 m above sea level, with an average distance between patches of 26 km. General Methods Blue tits adapt readily to breeding in nest boxes, which allows reproduction to be monitored. Frequent inspections of nest boxes allowed us to determine occupation by blue tits, date of clutch initiation for the different pairs, clutch sizes, and number of fledged young. In the study population, pairs breeding in open an degraded forest plots started later and had reduced hatching and breeding success (Arriero et al. 2006). No second clutches were observed in the population. For the purpose of this study we used two randomly selected nestlings of each brood. On day 15 after hatching (hatching date p 0), nestling tarsometatarsus length was measured to the nearest 0.01 mm with a digital caliper, and nestling body mass was recorded with an electronic scale to the nearest 0.1 g. A blood sample (∼50 mL) was obtained from the brachial vein and used both for hemoparasite detection in blood smears and stress protein analyses (see “Parasite Prevalence” and “Quantification of Stress Protein HSP60”). All nest boxes were cleaned after the breeding season.

min. Following the protocol by Merino et al. (1997), half of the symmetrical smear was scanned at #200 magnification to search for large extraerythrocytic hematozoa such as Filaria or Trypanosoma. In the other half of the smear, 20 microscope fields were examined at #400 magnification to detect intraerythrocytic parasites. Infections by Haemoproteus, Plasmodium, and Leucocytozoon can be detected by this method. Parasite prevalence was defined as the proportion of nests (for nest ectoparasites) or individuals (for hemoparasites) classified as positive for the presence of each parasite type. Quantification of Stress Protein HSP60 Circulating levels of HSP60 were quantified in the cellular fraction of the blood. Among the existing groups of HSPs (HSP90, HSP70, HSP60, and small HSPs) we focused on HSP60 because it has shown appealing associations with ecological variables in wild birds and in particular with parasitism (see Merino et al. 1998, 2002; Martıńez-Padilla et al. 2004; Toma´s et al. 2005). For the detection of HSP60, we used a Western blot protocol, with anti-HSP60 (1/1,000, cloneLK2, Sigma H-3524) as primary monoclonal antibody and a peroxidase-conjugated antimouse antibody (1/6,000, Sigma) as a secondary antibody (see Toma´s et al. 2004 for a full description of the method). Immunoreactivity of blots was estimated by densitometric quantification (Scion, Frederick, MD), and the results are expressed in arbitrary units (mean density of the bands # area). In order to control for interassay variation, a standard (pooled cellular fraction from several nestlings) was run in two wells in all blots. Levels of HSP60 were log10 transformed before analyses.

Parasite Prevalence The occurrence of hen fleas Ceratophyllus gallinae (Siphonaptera: Ceratophyllidae), blowfly larvae Protocalliphora spp. (Diptera: Calliphoridae), or mites Dermanyssus gallinae (Acari: Dermanyssidae) infesting blue tit nests was assessed as a categorical variable denoting infested versus uninfested nests. A brood was considered infested when mites, fleas, or blowflies were detected on the nest box, on nest material at the end of the breeding season, or in nestlings when handled. A semiquantitative index with three categories was also used to assess mite and flea loads. We scored as 0 nests where no mites or fleas were detected either on nest material or in nestlings when handled on day 15 after hatching. Nests with scattered mites or fleas (!100) were scored as 1, and nests with 100–500 mites or fleas were scored as 2. Blowfly larvae are common blood sucking ectoparasites of Holarctic birds (Bennett and Whitworth 1991) and probably the most harmful ectoparasites for blue tit nestlings, as reported by studies in similar latitudes (Hurtrez-Bousse`s et al. 1997; Simon et al. 2004). Blowfly loads were assessed by counting pupae capsules embedded in the nest material at the end of the breeding season. Parasite load was log10 transformed before analyses. For the detection of blood parasites, blood smears were fixed in absolute ethanol and stained with Giemsa (1/10 v/v) for 45

Molecular Sexing Molecular sexing of nestlings was conducted using primers P2 and P8, following Griffiths et al. (1998). Two homologous genes were amplified with this method: CHD1Z, occurring in both sexes, and CHD1W, occurring only in the W chromosome carried by females. The PCR products were separated on 1.5% agarose gel containing ethidium bromide and visualized under ultraviolet light. Samples taken from six breeding adults were correctly sexed using this method. Nine nestlings sexed from blood samples with this protocol were recaptured during the next breeding season as adults. In all cases, the sex determination using genetic markers agreed with that determined in the field based on plumage coloration and the presence of brood patch. Statistical Analyses Parasite prevalence in relation to habitat characteristics was analyzed using generalized linear models, working with the binomial distribution and the logit transformation as the link function. In the models, forest structure, year, and locality (nested within forest structure) were included as explanatory factors. Presence versus absence of the different parasites in

198 E. Arriero, J. Moreno, S. Merino, and J. Martıńez Table 1: Prevalence of parasites in the study population 2001

Blowflies Fleas Mites Leucocytozoon

2002

N

%

N

%

114 114 114 69

39.47 30.70 11.40 15.94

115 115 115 48

29.96 28.69 6.96 12.5

Note. Sample sizes indicate the total number of nests or broods examined each year.

nests (ectoparasites) or chicks (hemoparasites) was the binary dependent variable in each model. Individual variation in stress protein levels was examined using a mixed-model ANOVA where nest ID was considered a random effect. This design avoids pseudoreplication and allows for comparison among individual nestlings because covariation among nest mates is controlled as a random effect (Littell et al. 1996). To account for noncontrolled year or local effects, year and locality were included as random factors in the analysis. The design uses Type I sums of squares to test the significance of effects. Thus, the variables are entered in a stepwise fashion, controlling for all other factors that have been previously entered and ignoring those that have not yet been entered. Error terms are constructed using the Satterthwaite method of denominator synthesis (Satterthwaite 1946). This method finds the most appropriate linear combination of sources of random variation to construct the error term for testing the significance of each effect of interest. The analysis included the following fixed effects: habitat structure, hatching date, brood size, nestling sex, presence/absence of mites and fleas, blowfly loads, and prevalence of Leucocytozoon. Sample sizes varied depending on data analyses; not all the nests where the presence of parasites was determined could be controlled to obtain blood samples and morphological measurements of nestlings. The design is unbalanced because one of the forest plots could be monitored only in the breeding season of 2001 for logistical reasons. In our data set, there are no nestlings from the same pair breeding in consecutive years. All analyses were performed with Statistica (StatSoft). Mean Ⳳ SE is presented throughout. Results Habitat Characteristics and Parasite Prevalence A total of 229 nests were checked for the presence of ectoparasites. The most common ectoparasites in our study population were blowflies and hen fleas, while infestations by mites were less frequent (Table 1). Nests parasitized by blowflies hosted on average (9.97 Ⳳ 1.21) pupae, with a range of 1–54. Protocalliphora spp. loads were positively associated with brood size and hatching date (brood size: F1, 109 p 8.69, P p 0.004; hatching date: F1, 109 p 8.93, P p 0.003). Loads per nestling in par-

asitized nests ranged between 0.1 and 4.5 larvae per nestling, with an average of 1.34 Ⳳ 0.19. The occurrence of ectoparasites was examined in relation to habitat characteristics. Infestation by mites was not significantly associated with habitat characteristics. However, flea-infested nests were more frequent in mature forests than in young and open forests. The occurrence of blowflies was associated with young and cleared forests (Table 2; Fig. 1). Yet, blowfly larvae loads were not significantly associated with habitat characteristics when we controlled for hatching date and brood size (F1, 09 p 2.13, P p 0.147). No significant interannual variation was observed in parasite prevalence (Tables 1, 2). However, blowfly loads were higher in 2001 than in 2002 (F1, 109 p 7.19, P p 0.008). The occurrence of multiple infestations (11.3% of nests examined) was not significantly associated with habitat characteristics (x 32 p 1.747, P p 0.626). The results did not change when we used the semiquantitative index of mite and flea loads per nest. Thus, for simplicity, only the results concerning presence/absence are presented. Leucocytozoon majoris was the hemoparasite with the highest prevalence in the population (Table 1), although infection by Trypanosoma spp. was detected in one nestling. The prevalence of L. majoris was associated with habitat characteristics (Table 2). Higher prevalence was associated with mature forests, and no significant interannual variation was observed (Table 2; Fig. 1). Stress Response The analysis revealed a strong association between parasitic infections and circulating levels of stress protein HSP60 (Table Table 2: Prevalence of parasites in relation to habitat characteristics Source of Variation Blowflies: Habitat structure Year Locality Fleas: Habitat structure Year Locality Habitat # year Mites: Habitat structure Year Locality Leucocytozoon : Habitat structure Year Locality

x2

df

Log Likelihood

P

1 1 4

⫺74.232 ⫺73.002 ⫺65.718

4.299 2.461 14.567

.038 .117 .006

1 1 4 1

⫺63.429 ⫺62.837 ⫺58.707 ⫺55.897

22.981 1.184 8.261 5.621

!.001

1 1 4

⫺46.189 ⫺45.681 ⫺40.044

1.865 1.016 11.272

.172 .313 .024

1 1 4

⫺42.368 ⫺42.368 ⫺37.520

11.933 .0004 9.695

.276 .082 .018

!.001

.983 .046

Note. Generalized linear model was used, with infested versus uninfested as binomial dependent variable and logit as the link function.

Habitat, Parasites, and Stress Proteins in Growing Birds 199

Figure 1. Mean (ⳲSE) prevalence of different parasites in relation to habitat structure. Black bars p fleas, gray bars p mites, open bars p blowflies, stippled bars p Leucocytozoon.

3). Blowfly loads and Leucocytozoon infection were associated with higher levels of HSP60 (Table 3; Figs. 2, 3). However, HSP60 levels were not directly associated with habitat structural characteristics (Table 3). In addition, there was a significant interannual variation in circulating levels of stress proteins (Table 3). Hatching date, brood size, nestling sex, and the occurrence of fleas or mites were not significantly associated with circulating levels of HSP60 (Table 3). Discussion In a population of blue tits breeding in central Spain, we have shown that the occurrence of some of the most harmful parasites for avian hosts (Atkinson and van Riper 1991; Whitworth and Bennett 1992) is associated with forest structural characteristics. The role of habitat characteristics influencing offspring growth and development has generally focused in variation in food availability (Martin 1987; Ardia 2006). However, in populations of insectivorous birds in their preferred breeding habitat, food limitation during the nestling period might not be the primary restriction for reproduction (Tremblay et al. 2003). Other important selection pressures for growing birds, such as the impact of parasitism, can reduce offspring fitness, either directly or by interacting with food availability. Variation in parasite prevalence is a function of both adequate characteristics for the parasite (e.g., parasite-specific vector abundance) and the ability of the host immune system to ¯ clear infections (Frank 2002; Valkiunas 2005). Several previous studies have shown that habitat characteristics can predict variation in parasite prevalence across species at either broad (Peirce 1981; Scheuerlein and Ricklefs 2004; Mendes et al. 2005) or small geographical scales (Greiner et al. 1975; Garvin and Remsen 1997; Tella et al. 1999). However, intraspecific differences in parasite prevalence within the same region and habitat type have normally been attributed to host traits, including

disease resistance ability, age, breeding cycle, and behavioral antiparasite mechanisms (reviewed in Møller 1997). By studying altricial nestlings that had a naive immune system (Apanius 1998) and were confined to the nest, we were able to show that forest structural characteristics and local effects, presumably associated with vector abundance, can determine the incidence of newly acquired parasitic infections. Hence, habitat features may indirectly influence the physiological state of growing nestlings through parasite infections. Environmental conditions such as air temperature and moisture can vary to a great extent with vegetation structure (Cody 1985; Hunter 1999). Thus, the characteristics of the forest can determine the abundance of arthropod vectors or ectoparasites. Hematophagous blackflies from the family Simuliidae (Diptera) are the best known vectors of Leucocytozoon and require running water for the immature stages (Greiner et al. 1975; Desser and Bennett 1993). Therefore, habitat features can determine the abundance of this water-dependent insect group and thus the incidence of the hempoarasite infection with the highest prevalence in European blue tits (Merino et al. 1997; Scheuerlein and Ricklefs 2004). Blood-sucking fleas can act as vectors of some hemosporidian parasites (Allander and Bennett 1994), and the occurrence of fleas was also associated with areas with mature vegetation. In addition, mature forest plots maintained higher breeding densities (Arriero et al. 2006), which may favor parasite transmission among adults. In contrast, the occurrence of blowflies, which feed solely on nestling blood, was higher in heavily managed forest plots characterized by young and degraded vegetation. Unlike other ectoparasites such as mites and fleas, adult blowflies are freeleaving insects and do not remain in the nest material from one breeding season to the next (Bennett and Whitworth 1991). Microclimatic conditions of these open and probably dryer forest plots might favor the occurrence of blowflies. However, infestation by blowfly larvae is tightly linked to nestling hatching date (Merino and Potti 1995; this study) and brood size (Eeva et al. 1994; this study), which may be associated with habitat characteristics. In our study population, the onset of breeding is delayed in areas of open and degraded vegetation (Arriero et al. 2006). Presumably, adult flies lay eggs on avian nests at chick hatching (Bennett and Whitworth 1991). Thus, the delay in the onset of reproduction coinciding with warmer climate favorable for flying insects may explain the higher impact of blowfly infestation in degraded habitats. The incidence of mites in our population was low compared to those of other populations of blue tits in central Spain (Toma´s et al. 2007) and was not significantly associated with forest characteristics. Nevertheless, none of the parasites found in this study is unique to either young or mature forest habitats, but most occur in both types of habitats. Vegetation characteristics and humidity are probably more important determinants of the abundance of arthropod vectors and ectoparasites than climatic factors at a wider scale since no interannual variation in the prevalence of parasites was observed in our study. However, weather conditions can deter-

200 E. Arriero, J. Moreno, S. Merino, and J. Martıńez mine the severity of the impact of parasites on offspring fitness (de Lope et al. 1993; Merino and Potti 1996). Time-dependent mortality and long-term fitness consequences resulting from parasitism can act as important selection pressures on nestling growth and development (Clayton and Moore 1997; Fitze et al. 2004). Infections and other general responses to disease, such as fever or inflammation, may cause the induction of HSP60 in nestlings (Feder and Hofmann 1999). Previous studies have shown elevated levels of HSP60 associated with parasitic infections in wild birds (e.g., Merino et al. 1998; Martıńez-Padilla et al. 2004). Thus, an upregulation of HSPs may be a physiological strategy to reduce the impact of parasitism in nestlings. The association between Leucocytozoon infection and HSP60 levels may be a consequence of fever caused by the infection or the oxygen demand resulting from the destruction of infected blood cells (Feder and Hofmann 1999). Besides, stress proteins might be produced by host cells as a direct consequence of the presence of the parasite in the blood. Ectoparasite infestations can also activate a stress response mediated by HSPs. Blood-sucking parasites such as mites, fleas, and blowfly larvae secrete anticoagulant substances during their blood meals and also transmit other biochemical substances that can activate inflammatory responses (Wakelin and Apanius 1997). However, only blowfly infestation was associated with elevated levels of stress proteins in our study. The loss of a significant amount of blood associated with blowfly infestation (Simon et al. 2004) might be a more important determinant of the activation of the stress response than biochemical compounds or the cutaneous immune reaction caused by blood sucking. Blue tit nestlings parasitized by Protocalliphora larvae suffer from reduced thermogenic and metabolic capacities as a result of reduced hematocrit (Simon et al. 2004), which could explain the elevated levels of HSP60 found in our study. The oxygen carrying capacity may be reduced by up to 39% as a consequence of Protocalliphora infestation (Simon et al. 2004), and the oxygen deficit may trigger the expression of HSPs (Feder and Hofmann 1999). Flea infestation has been shown to impair offspring quality and survival in other passerine species, including

Figure 2. Mean (ⳲSE) HSP60 levels in relation to Leucocytozoon infection (infected p 20,010.32 Ⳳ 1,678.09, N p 22; uninfected p 14,993.86 Ⳳ 643.72, N p 188). Back-transformed HSP60 levels are expressed in arbitrary units (optical density # area).

the closely related great tit (Parus major; Richner et al. 1993). However, flea infestations generally represent a less significant loss of blood for the host, and the negative effects might not be detectable at the nestling period, showing up only in adults later in their life. Yet, the subtle effects of some ectoparasites may also play an important role in the trade-off between current and future reproduction (Møller 1997). Sexual differences in the impact of parasitism on nestling growth and survival have been reported for other passerine species (Potti and Merino 1996; Badyaev et al. 2006). In our study population, male and female nestlings differed in morphology and carotenoid-based plumage coloration (Arriero and Fargallo 2006). However, no significant differences between males and females were detected in circulating levels of HSP60. The physiological stress response mediated by HSPs seems to be independent of sex at that age, although sexual differences

Table 3: Mixed-model ANOVA of circulating levels of stress protein (HSP60) in nestlings

Habitat structure Leucocytozoon Protocalliphora load Fleas Mites Hatching date Brood size Sex Locality Year Nest

Factor

df (Effect, Error)

SS

F

P

Fixed Fixed Fixed Fixed Fixed Fixed Fixed Fixed Random Random Random

1, 6.08 1, 76.61 1, 24.46 1, 10.30 1, 32.46 1, 2.25 1, 9.91 1, 30.30 4, 81.60 1, 82.24 84, 79

.189 .416 .749 .378 .027 .015 .010 .015 .450 1.628 7.509

1.372 5.785 6.565 2.838 .251 .055 .076 .217 1.220 17.43 3.147

.285 .019 .017 .122 .620 .834 .788 .645 .309 !.001 !.001

Habitat, Parasites, and Stress Proteins in Growing Birds 201 00787/BOS (Ministerio de Ciencia y Tecnologıá) to J. Moreno. M. Romero-Pujante, J. A. Fargallo, J. J. Sanz, A. Martin, and A. Nicolau helped with the fieldwork. El Ventorrillo field station provided infrastructure and logistical support. Comunidad de Madrid authorized the fieldwork in the study area and the capture and ringing of birds. Comments by three anonymous reviewers greatly improved earlier versions of the manuscript. E.A. was supported by a fellowship from El Ventorrillo–Consejo Superior de Investigaciones Cientı´ficas. During final analyses and writing, support to E.A. was provided by a postdoctoral fellowship from the Spanish Ministry of Education and Science and by the Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 7103, France. Literature Cited Figure 3. Levels of HSP60 (optical density # area ) in relation to Protocalliphora load on nests. Linear regression based on back-transformed variables (y p 14,162.71 ⫹ 272.25x).

have been reported in adult birds of other species (Merino et al. 2002). Parasitic infections, in combination with other stressful conditions such as food limitation or sibling competition, might reveal sex-biased differences in strategies to cope with physiological stress (Martıńez-Padilla et al. 2004). Levels of HSP60 have been also associated with nutritional stress in nestlings of other passerine species (Merino et al. 1998; Moreno et al. 2002). Yet, in our study population, food availability seemed not to be limiting during the nestling period, since nestling body size before fledging was not significantly associated with habitat characteristics of the breeding territories (Arriero et al. 2006). Not surprisingly, then, brood size did not predict variation in levels of HSP60 in our population. Physiological techniques are increasingly used to assess the conservation status of populations (reviewed in Wikelski and Cooke 2006). Other physiological indicators of stress, such as heterophil/lymphocyte ratio and plasma corticosterone, have been shown to reflect variation in forest features (Eeva et al. 2000; Suorsa et al. 2003, 2004). Our study shows that stress proteins can be used as an integrated measurement of offspring rearing conditions as well because the association between habitat characteristics and incidence of parasitism can be tracked through the expression of stress proteins. The opposing effect of habitat structure on the prevalence of the most deleterious parasites for the species might be obscuring a direct association between habitat features and stress response. Future studies should integrate different estimates of physiological stress response to allow better understanding of different response mechanisms and consequences of habitat alteration for wild populations. Acknowledgments The study was financially supported by projects 07M/0137/2000 (Comunidad de Madrid) to L. M. Carrascal and CGL2004-

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