Nest construction rate and stress in female Pied Flycatchers Ficedula hypoleuca

ACTA ORNITHOLOGICA Vol. 43 (2008) No. 1 Nest construction rate and stress in female Pied Flycatchers Ficedula hypoleuca Juan MORENO 1*, Javier MARTÍNEZ 2, Consuelo CORRAL 1, Elisa LOBATO 1, Santiago MERINO 1, Judith MORALES 1, Josué MARTÍNEZ-DE LA PUENTE 1 & Gustavo TOMÁS 1 1 Department of Evolutionary Ecology, National Museum of Natural Sciences-CSIC, J. Gutiérrez Abascal 2, E-28006 Madrid, SPAIN 2 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Alcalá, E-28871 Alcalá de Henares, SPAIN *Corresponding author, e-mail: jmoreno@mncn.csic.es Moreno J., Martínez J., Corral C., Lobato E., Merino S., Morales J., Martínez-de la Puente, Tomás G. 2008. Nest construction rate and stress in female Pied Flycatchers Ficedula hypoleuca. Acta Ornithol. 43: 57 64. DOI 10.3161/000164508X345338 Abstract. Nest building effort has received scant attention in the literature although it may involve costs which can be detected as physiological stress. We prolonged nest construction effort in a population of Spanish Pied Flycatchers by removing nests from nest-boxes and forcing females to build a second nest. In comparison with control nests, the experimental females had to work for longer periods and accumulate more nest material, but nest construction rates (g of nest material per day of construction) were not affected. There was a positive association of clutch mass with nest construction rate. To measure physiological stress, we captured females shortly after laying to obtain blood samples for heat-shock protein quantification. Heat-shock proteins quantify stress at cell level. The level of HSP60 in peripheral blood was positively associated with total nest construction rate (including second nests for experimental females), but not with laying date, clutch mass or experimental treatment. A third of the variation in the HSP60 level was explained by the nest construction rate. Fast nest builders are physiologically stressed, suggesting that the nest construction rate may constitute an index of female physiological performance. Key words: nest construction rate, physiological stress, Pied Flycatcher, Ficedula hypoleuca, stress proteins, nest building, female performance Received March 2008, accepted May 2008 INTRODUCTION Nest construction has been traditionally neglected as a potential source of life history tradeoffs (Hansell 2000). There is evidence that in certain species, nest building implies severe energetic costs (Withers 1977, Lens et al. 1994, but see Stanley 2002) and considerable time allocation (Hotta 1994, Nores & Nores 1994). In fact, it has been repeatedly proposed that builders in certain species may signal quality or parental disposition also through construction activities, and that their partners may respond by allocating more reproductive effort as proposed by the differential allocation hypothesis (Moreno et al. 1994, Soler et al. 1996, 1998a, 2001, de Neve & Soler 2002, Szentirmai et al. 2005). Soler et al. (1998b) have proposed that nest building activity could be used as a sexually selected display, allowing each sex to obtain reliable information about the condition of its mate. In Blue Tits Cyanistes caeruleus, where females build nests, there is evidence that nest size is related to female health and that building large nests has costs in terms of reduced immunocompetence (Tomás et al. 2006). Female Buff-breasted Wrens Thryothorus leucotis that build dormitory nests at higher rates suffer costs in terms of survival (Gill & Stutchbury 2005), again indicating the potential costs of nest building. Thus, nest size or nest construction rate could function as post-mating sexually selected traits also for females (Soler et al. 1998b). Males may be selected to respond parentally to female nest building activity in those

58 J. Moreno et al. species where females are the main nest builders (Borowiec et al. 2006). The assumption in these studies is that nest construction is in some way costly for the builders, thus signaling some quality trait associated with nest building performance. If this assumption is true, costs of nestbuilding could be expressed as physiological stress. Animals experience adverse conditions during their lifetime in terms of resource acquisition or demand that may elicit behavioral and physiological adjustments called stress responses (Buchanan 2000). The differential synthesis of heat shock proteins (HSP) has been increasingly used in recent years as an indicator of stress in wild animal populations (Srrensen et al. 2003). Ecological factors such as parasitism, infection, energetic exertion or population density may also elicit HSP synthesis and mobilization in natural conditions as shown in recent studies (Srrensen et al. 2003). Measuring HSP levels may therefore constitute an adequate way of estimating natural stress in the wild (Buchanan 2000, Herring & Gawlik 2007). Variation in levels of HSPs in important organs and tissues may be detected in peripheral blood as circulating blood cells may be stimulated to increase HSP synthesis. In animals with nucleated blood cells such as birds, heightened HSP levels may thus be estimated non-destructively through blood sampling (Herring & Gawlik 2007). In wild birds, heightened HSP levels have been linked to ectoparasite infestation (Merino et al. 1998, 2001), haemoparasite infection (Merino et al. 2002, Morales et al. 2004, Tomás et al. 2005), impaired growth (Moreno et al. 2002), heightened competition in the nest (Martínez-Padilla et al. 2004, Blanco et al. 2006), fasting incubation (Bourgeon et al. 2006), intense parental effort (Merino et al. 2006), delayed moult (Morales et al. 2007) depressed immunocompetence (Morales et al. 2006), experimentally induced immune responses (Sanz et al. 2004) and higher song complexity (Garamszegi et al. 2006). Several of these studies manipulated stressors experimentally and detected significant effects on stress protein levels (Merino et al. 1998, Sanz et al. 2004, Tomás et al. 2005, Merino et al. 2006). Thus, not only extreme ambient conditions like the heat stress that gave these proteins their name, but normal functions like breeding or growth elicit variation in avian HSP levels. In comparison to corticosterone, stress proteins may be a more appropriate metric for detecting longterm effects because it takes several hours to show up in blood cells and remain there longer than corticosterone (Herring & Gawlik 2007). As stressed by Herring & Gawlik (2007), stress proteins increase the options available to avian ecologists for understanding how avian species respond to changes in the environment. In the present study we test the possible stressful effects of nest building activity. We have addressed the possible effects of nest construction on female physiological stress as inferred from levels of HSP60 in blood in a small songbird where females are the main nest builders (only 25% of males take any part in this activity, Martínez-de la Puente et al. submitted), the Pied Flycatcher (Curio 1959, Lundberg & Alatalo 1992). Nest construction activity may be estimated through its rate (de Neve & Soler 2002, Gill & Stutchbury 2005), through its duration (Szentirmai et al. 2005) or through its product in terms of mass collected (Soler et al. 2001, Szentirmai et al. 2005) or associated evidence (Borowiec et al. 2006). To separate construction rate, duration and nest size, we conducted an experiment in which some nests were removed at completion and females had to build a second nest, while control nests were only weighed at completion and placed back. While this manipulation prolongs nest-building activity and may have effects on final nest size, it does not necessarily change the nest construction rate. Females were captured one week after clutch completion to estimate condition and stress. The hypothesis tested is that if duration of nest-building or amount of nest material carried has any bearing on costs, treatment should be related to physiological stress in females. Alternatively, either nest construction rate could be related to stress independently of treatment or no associations of stress with any nest-building parameter would be detected, which would negate substantial costs of nest-building activity. METHODS The study was conducted in 2006 in a population of Pied Flycatchers breeding in nest-boxes close to the village of Lozoya, central Spain (40 58 N, 3 48 W). The study area is a montane Pyrenean Oak Quercus pyrenaica forest on an eastfacing slope at 1500 m elevation, where 100 nestboxes were erected in 2001. Pied Flycatchers occupy approximately half of the nest-boxes each year. Nest-boxes were checked daily from arrival of the first males to the study area (April 15) in order to detect the first indications of nest construction by

Stress proteins and nest construction 59 Pied Flycatchers, and the process of nest construction was followed through daily visits until nests showed the rounded and closely knit nest cup indicating completion, when they were extracted from the nest-box, weighed to the nearest 0.1 g on a portable electronic balance, and either placed back in the nest-box or removed. The same observer made the subjective decision about nest completion, so any error of appreciation was probably systematic (for obvious reasons nest removal can only be carried out some time before laying, which means as soon as there is a cup in the nest). Nests are sufficiently compact to allow removal and weighing without deterioration. Control and experimental treatments were allocated to 48 nests (24 to each treatment) randomly during each day on which completed nests were found. In all experimental nest-boxes, nest construction was resumed immediately (next two days) after removal of the first nest. Take-over by another female in this short time and initiation of a new nest remains possible but not plausible due to the stiff competition among females for nestboxes in the study area (which may be lethal, J. Moreno pers. obs.). We assume that resumption of nest-construction in such a short interval implied a second attempt by the same female. The second nests in experimental nest-boxes were also weighed at completion. All nests included in the study were dry on weighing and showed a well constructed nest cup (as subjectively determined by the observer, see above). Thus, all nest weights refer to measurements on the first day when we estimated the nest to be complete. The day of clutch initiation was detected in all cases and eggs were weighed on the day of laying with a portable electronic balance. One week after clutch completion, most incubating females were captured in the nest-box during daytime without traps as they usually sit very tight on the eggs. We did not capture them at an earlier stage, given the increased risk of nest desertion. They were banded if necessary, identified, weighed to the nearest 0.25 g with a PESOLA spring balance, and we measured their tarsus length with a digital caliper to the nearest 0.01 mm. Female mass was corrected by size by using tarsus length as a covariate in analyses (residuals could not be used as mass was not significantly related to tarsus length). Condition will be expressed as mass divided by the cube of tarsus length. Blood was sampled through venipuncture of a brachial vein and collected in an 85 microliter heparinized capillary tube, from which it was immediately transmitted to a plastic vial with lock. Many females continued incubating after being placed back on the nest. Blood was centrifuged in the field with a portable centrifuge and separated into plasma and cellular fractions. The cellular fraction was frozen on the day of sampling at -80 C until HSP analysis (Tomás et al. 2004). Estimates of HSP60 levels were using made Western Blot following Merino et al. (2002), Tomás et al. (2004) and Merino et al. (2006). We have used general linear models in the STATISTICA software package for statistical analyses, after checking that no pairs of variables exceeded the usual collinearity standard of r 0.70. As not all females could be captured and not all nests fulfilled conditions for inclusion (see above), sample sizes differ for different analyses. RESULTS There was no difference in clutch size, clutch mass, female condition, or original nest size according to treatment (Table 1). The experimental treatment resulted in prolonged nest construction periods, larger total nest masses, and smaller final incubation nests. We also observed delayed initiation of laying and shorter intervals between the end of construction and laying in experimental nests (Table 1). However, no effect of treatment on nest construction rate of second nests or construction rate during the whole construction period was found (Table 1). The experiment thus allows us to separate nest construction duration, product and rate as possible determinants of stress, as it changed the first two but not the last. Experimental females building larger original nests, also built larger second nests (r 12 = 0.64, p = 0.014) (Fig. 1), although second nests were clearly smaller (Table 1). The rate of construction of second nests was not significantly higher than the rate of construction of original nests for experimental nest-boxes (second nests 3.3 ± 0.3 g/day versus original nests 4.2 ± 0.7 g/day, n = 14, paired t = 1.3, p = 0.20). Construction rates of original nests were significantly associated with rates of second nests only for the 11 females with original rates less than twice the mean value (r 9 = 0.62, p = 0.04). The three females building original nests at the highest rates dropped markedly in performance for second nests, with construction rates at half the original value. To test the dependence of HSP60 levels on prior female reproductive effort we established a GLM model with experimental

60 J. Moreno et al. Table 1. Effects of experimental removal of nests after completion and subsequent construction of a second nest (values are means ± SE and sample sizes in parentheses). Not all females could be captured nor all nests properly weighed which explains differences in sample sizes. Control Experimental F p Laying date (1 = April 1) 43.1 ± 0.7 (24) 45.5 ± 0.8 (20) 4.97 0.031 Clutch size 6.1 ± 0.1 (24) 5.9 ± 0.1 (20) 1.46 0.23 Clutch mass (g) 10.0 ± 0.2 (24) 9.8 ± 0.2 (19) 0.45 0.5 Interval between end of construction and laying (days) 5.4 ± 0.4 (22) 2.8 ± 0.4 (20) 17.8 < 0.001 Female condition x 100 (g/mm 3 ) 0.31 ± 0.01 (19) 0.30 ± 0.01 (18) 0.47 0.5 Original nest (g) 24.2 ± 1.3 (22) 27.3 ± 1.5 (16) 4.1 0.13 Final nest (g) 24.2 ± 1.3 (22) 17.0 ± 1.1 (18) 24.1 < 0.001 Sum of nests (g) 24.2 ± 1.3 (22) 44.2 ± 2.1 (14) 54.1 < 0.001 Total construction period (days) 8.6 ± 0.6 (22) 13.4 ± 0.7 (20) 26.9 < 0.001 Construction rate original nests (g/day) 3.15 ± 0.37 (22) 3.97 ± 0.43 (16) 2.1 0.16 Construction rate final nests (g/day) 3.15 ± 0.37 (22) 3.53 ± 0.27 (18) 1.1 0.3 Construction rate all nests (g/day) 3.15 ± 0.37 (22) 3.65 ± 0.35 (14) 1.5 0.22 treatment as factor, and female mass and tarsus length, laying date of the first egg, clutch mass and total nest construction rate as covariates. Total nest construction rate was calculated as the sum of masses of nests divided by total number of days elapsed in construction. This variable was positively associated with the rates of construction of original nests for all nest-boxes (r 34 = 0.93, p < 0.001) and of second nests for experimental nestboxes (r 12 = 0.65, p = 0.011). As egg synthesis may overlap with the end of nest building, especially for experimentally delayed nests, the interval between end of nest construction and the laying of the first egg was included as another covariate in the model. The model explaining HSP60 levels in incubating females included only total nest construction rate as significant and explained 31% of the variation (Total model F 7,31 = 3.4, p = 0.008, Table 2). Females with a faster nest construction exhibited higher HSP60 levels while incubating (r 29 =0.59, p < 0.001)(Table 2, Fig. 2). Polynomial, logarithmic or exponential functions did not noticeably increase the adjusted r 2 which fluctuated between 0.30 and 0.33. Treatment showed no association with HSP60 level (Table 2). Mass was not related to HSP60 level when correcting for tarsus length (Table 2). We found no association of HSP60 level with laying date, clutch mass or interval between the end of construction and laying (Table 2). Total nest construction rate was positively associated with clutch mass (r 32 = 0.52, p = 0.001)(Fig. 3), but not with laying date 24 250 New nest mass (g) 22 20 18 16 14 12 New nest mass (g) 10 8 6 15 20 25 30 35 40 45 50 Original nest mass (g) (g) Fig. 1. Association between mass of original and final nests built by females in the experimental treatment. HSP60 level HSP60 level 240 230 220 210 200 190 180 170 1 2 3 4 5 6 7 Total Total nest construction rate (g/day) Fig. 2. Level of HSP60 (expressed in arbitrary units) in peripheral blood in relation to nest construction rate (g nest mass per day of building).

Stress proteins and nest construction 61 Table 2. Results of a general linear model analysis explaining the level of the stress protein HSP60 in female blood during incubation (beta coefficients with SE are only presented for covariates). Beta SE F p Treatment 1.93 0.17 Laying date -0.20 0.17 1.52 0.23 Clutch mass -0.07 0.18 0.16 0.69 Interval between end of construction and laying 0.19 0.18 1.06 0.31 Female mass <0.01 0.15 < 0.01 0.97 Female tarsus length -0.05 0.18 0.09 0.76 Construction rate all nests 0.72 0.21 12.1 0.0015 (r 34 = 0.03, p = 0.86). The association of HSP60 level with clutch mass was significant when only considering control nests (r 20 = 0.46, p = 0.031). Original nest size and final nest size were not associated with clutch mass (p > 0.10). DISCUSSION Nest construction in the Pied Flycatcher is carried out mainly (Martínez-de la Puente et al. submitted) or exclusively (Curio 1959) by females. The average nest construction period of 8 9 days in control nests is longer than the average of 5 days reported by Curio (1959) for a German population. Possible differences in nest design and general life history between populations may explain the longer nest construction periods in our population. Curio (1959) reports that females may visit the nest-box with nesting material up to 30 times/h. These high rates of nest visits may imply important energy costs, although this has not Clutch mass (g) 13 12 11 10 Clutch mass (g) 9 8 7 1 2 3 4 5 6 7 Total Total nest construction rate rate (g/day) (g/day) Fig. 3. Association between clutch mass and total nest construction rate (g nest mass per day of building) for all nests. been quantified. Construction rates in our population show ample variation, with a six-fold difference between range extremes. This suggests that inherent variation in female building performance could be involved. If construction rates were not constrained by performance, we should expect that females in the experimental group would considerably increase construction in order to avoid a delay in their breeding schedule. However, some of them even slowed down construction rates. Speeding up was only detectable in the time taken between end of construction and laying and in a reduction in final nest size in experimental nest-boxes. That the interval between construction and laying did not affect physiological stress suggests that experimental females did not suffer an additional stress due to producing eggs while still constructing final nests. The results show that nest construction rate was positively associated with stress protein level of incubating females, while neither the duration of the activity nor the amount of nest material show such an association. In fact, treatment was totally unrelated to stress, and it did not affect construction rate. This is one of the few studies to attempt to experimentally alter nest construction costs by inducing females to build a second nest after having completed the first (but see de Neve & Soler 2002). Nest size was correlated with female health status in Blue Tits (Tomás et al. 2006), suggesting that only females in good health can build large nests. In our study, females building at higher rates lay heavier clutches but this is not the case for females building larger nests, either originally or experimentally induced. Nest size is necessarily positively related to construction rate in a context of time limitation, and may be used as a proxy for nest construction rate in certain situations (de Neve & Soler 2002). In this scenario, high quality individuals would build fast, constructing large nests in the limited time period available. Mates may judge quality from observing the activity or the result of it (de Neve & Soler 2002). Evidence in Black Wheatears Oenanthe leucura (Soler et al. 1996) and Magpies Pica pica (de Neve & Soler 2002) reveal that it is the activity itself and not its product that affects investment decisions by mates in certain contexts in which the size of the construction is not reliable or time is limited. It should be stressed here that all these studies deal with already mated pairs, and that the signal can only alter parental investment in common progeny (Sheldon 2000). Females are consistent with respect to nest size

62 J. Moreno et al. within seasons as shown by the positive correlation between first and second nests in the experimental treatment (see also de Neve & Soler 2002 for Magpies). But again, this may be only the expression of consistency in construction rate in a scenario of time limitation. Rate is consistent for low and average performers in the present study, while extremely high rates of building initial nests lead to decreased performance when building second nests. This again implies some cost of high construction rates, as females stressed by high initial rates would not be able to increase stress levels further. Pied Flycatchers are time constrained due to a short breeding season induced by their migratory habits and the need to synchronize their breeding season with seasonal food peak (Sanz et al. 2003), so they could be willing to pay the costs of fast nest construction. Not all females may be able to incur these costs, as evidenced by Blue Tit females in poor health (Tomás et al. 2006). So nest building capacity could in fact be another indicator for males of their mate s condition and performance as proposed by the nest building as signal hypothesis (Soler et al. 1998b, 2001) as applied to species where females are the main builders and males collaborate in breeding tasks. An alternative interpretation of the strong correlation found could be that already stressed females would be speeding up nest construction as an expression of a general fast-breeding strategy. HSP60 levels would only express a general disposition to breed as fast as possible and not the cost of nest building. However, the fact that no other variable included in the model like laying date or interval between nest building and laying was associated with HSP60 level makes this possibility less plausible. A key finding is that nest construction effort does not impact on subsequent egg production. Thus, treatment did not affect clutch size or clutch mass, and nest construction rate was in fact positively associated with clutch mass for the whole sample of nests and for control nests only. Thus, there is apparently no trade-off between nest construction effort and egg production effort. However, nest construction rate was not affected by the experimental treatment, and only a successful manipulation of this variable may reveal its life history implications. Our correlational study only shows that females capable of building nests fast are also those laying heavy clutches, a typical result of studies of individuals with differences in resource access (van Noordwijk & de Jong 1986). However, it can be concluded that neither duration of the process nor total amount of nest material carried have probably any life history implications as they did not affect either egg production or female physiological stress. To conclude, stress protein levels in incubating female Pied Flycatchers suggest that previous nest construction effort may be stressful at the level of cell function. Individuals may differ with respect to the capacity to resist this stress, so nest building capacity as expressed by nest construction rate could constitute an index of female physiological performance. ACKNOWLEDGEMENTS The present study was financed by projects CGL2004-00787/BOSA and CGL2007-61251 to J. Moreno and CGL2006-14129-C02-01 to S. Merino and J. Martínez. Consejería de Medio Ambiente (Comunidad de Madrid) authorized the ringing and blood sampling of birds. R. Ruiz de Castanéda helped greatly with field work. E. Lobato and J. 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Tomás G., Merino S., Martínez J., Moreno J., Sanz J. J. 2005. Stress protein levels and blood parasite infection in blue tits: a medication field experiment. Ann. Zool. Fenn. 42: 45 56. Tomás G., Merino, S., Moreno J., Sanz J. J., Morales J., García- Fraile S. 2006. Nest weight and female health in the Blue tit (Cyanistes caeruleus). Auk 123: 1013 1021. Van Noordwijk A. J., de Jong G. 1986. Acquisition and allocation of resources: their influence on variation in life history tactics. Am. Nat. 128: 137 142. Withers P. C. 1977. Energetic aspects of reproduction by the Cliff Swallow. Auk 94: 718 725. STRESZCZENIE [Budowa gniazda a stres fizjologiczny samic muchołówki żałobnej] Badania przeprowadzono w populacji muchołówki żałobnej gniazdującej w skrzynkach lęgowych w środkowej Hiszpanii. Po rozpoczęciu przez ptaki budowy gniazd, podzielono je na dwie grupy. Części usuwano gotowe gniazda zanim nastąpiło składanie jaj, zmuszając samice do ponownej budowy. W ten sposób w grupie eksperymentalnej ptaki pracowały nad budową gniazd dłużej i przynosiły więcej materiału niż w kontroli. Gniazda u obu grup (w grupie eksperymentalnej zarówno pierwsze tj. usuwane jak i zbudowane ponownie) były ważone, znano także daty rozpoczęcia i zakończenia budowy. Określano czas przystępowania do lęgów, wielkość zniesienia i jego masę. Aby zmierzyć stres fizjologiczny (na poziomie komórkowym) związany z budową gniazda łapano samice w pierwszym tygodniu wysiadywania jaj, pobierano im kroplę krwi i oznaczono w niej białka szoku cieplnego HSP60. Samice były także ważone i mierzone (skok), aby oszacować ich kondycję.

64 J. Moreno et al. Nie stwierdzono różnic w grupach eksperymentalnej i kontrolnej w wielkości zniesienia, czasie przystępowania do lęgu czy kondycji samic, tempo budowy gniazd było podobne, choć drugie gniazda były mniejsze niż gniazda grupy kontrolnej (Tab. 1). Samice budujące większe gniazda pierwsze także drugie gniazda budowały większe (Fig. 1). Stwierdzono istotny związek między tempem budowy gniazda a wielkością zniesienia (Fig. 3). Poziom HSP60 był związany z tempem budowy gniazda tj. ilością materiału przynoszonego na dzień (Fig. 2), a nie z eksperymentem (usuwaniem/pozostawianiem gniazda), czy wielkością zniesienia (Tab. 2). 1/3 zmienności w poziomie białek HSP była wyjaśniana przez tempo budowy gniazda. Osobniki szybko budujące gniazda znajdują się w silniejszym fizjologicznym stresie, co wskazuje, ze tempo budowy gniazda może być wskaźnikiem kondycji fizjologicznej samicy. T. Cofta