Journal of Avian Biology 44: 521530, 2013 doi: 10.1111/j.1600-048X.2013.00181.x 2013 The Authors.

. Journal of Avian Biology 2013 Nordic Society Oikos Subject Editor: Staan Bensch. Accepted 15 May 2013

Haemosporidian parasitism in the blackcap Sylvia atricapilla in relation to spring arrival and body condition
Diego Santiago-Alarcon, Raeann Mettler, Gernot Segelbacher and H. Martin Schaefer
D. Santiago-Alarcon (diego.santiago@inecol.edu.mx), R. Mettler and H. M. Schaefer, Dept of Ecology and Evolutionary Biology, Faculty of Biology, Univ. of Freiburg, Freiburg, Baden-Wrttemberg, Germany. Present address of DSA: Biologa y Conservacin de Vertebrados, Inst. de Ecologa A.C., Xalapa, MX-91070 Veracruz, Mxico. G. Segelbacher, Dept of Wildlife Ecology and Management, Univ. of Freiburg, Freiburg, Baden-Wrttemberg, Germany.

Parasites can exert strong selection on hosts. Throughout the year migrants are exposed to dierent sets of parasites, which may aect life history traits such as migratory schedules. Here, we studied the relationship between parasite infection and arrival date of blackcaps Sylvia atricapilla to their breeding grounds in Germany throughout a period of six years (20072012). We used two data sets, one that included all blackcaps and one that included only recaptured birds. We assesed whether parasites inuence spring arrival to breeding grounds, and for the recaptured data set, we analysed temporal variation in parasitism (i.e. infection status and parasitaemia) throughout the breeding season. We used both microscopy and PCR (a fragment of 479 bp of the mtDNA cyt b) to determine haemosporidian infection. Blackcaps were mostly infected with Haemoproteus parabelopolskyi (lineages SYAT01 and SYAT02). Infection status, but not parasitaemia, was constant through time for individual birds; meaning that once a bird is infected, it most likely will retain the infection for life. We found that infection by haemosporidian parasites has no relationship to arrival date in this blackcap population; however, infection by H. parabelopolskyi has a marginally signicant eect on arrival date of recaptured blackcaps, somewhat delaying their arrival to breeding grounds. Birds captured later in the season were more likely to be infected than those from early spring, and parasitaemia was frequently lower in birds captured earlier in the season compared to those captured later (summer).

Parasites are selective agents on hosts (Cornell 1974, Piersma 1997) and in many instances are an essential component in the dynamics of ecosystems (Laerty et al. 2008). Avian haemosporidians are intracellular parasites from the genera Plasmodium, Fallisia, Haemoproteus and Leucocytozoon that can have negative tness eects on their hosts, such as high mortality in Hawaiian endemic birds (Atkinson et al. 2000, Yorinks and Atkinson 2000), lower reproductive output, lower survival, and hypertrophy of internal organs in several passerine species (Marzal et al. 2005, Palinauskas et al. 2008, Martnezde la Puente et al. 2010). However, there can also be negligible health eects (i.e. clinical signs) on some bird species heavily infected with haemosporidians (SantiagoAlarcon et al. 2012), such as temporary weight loss during the peak of the infection (Valkinas et al. 2006). Hence, haemosporidian health eects vary among bird species, going from lethal to sub-clinical symptoms (Valkinas 2005). Long distance movement exposes migratory birds to broader and dierent sets of parasites compared to residents (Waldenstrm et al. 2002, Garvin et al. 2006, Hellgren et al. 2007). Parasitism is then expected to be more costly

for migrants, due in part to the high energy requirements of migration (Newton 2008). For example, blood parasites aect the choice of migratory travel routes in red knots Calidris canutus (DAmico et al. 2008) and delay the onset of spring migration in wood-warblers Dendroica spp. (DeGroote and Rodewald 2010). However, blood parasites did not aect the distance traveled in sharp-shinned hawks Accipiter striatus (Smith et al. 2004) and the fuel deposition rate in blackcaps Sylvia atricapilla (Arizaga et al. 2009). Moreover, Haemoproteus prognei infected purple martins Progne subis have higher reproductive success and arrive earlier to breeding grounds compared to uninfected individuals (Davindar and Morton 1993, see also Marzal et al. 2008). These studies suggest that parasite pressure is not homogeneous across dierent migratory species, which partly depends on the parasite group under study (Rtti et al. 1993). Migratory species can aid the geographic range expansion of parasites, aid in the (re)emergence of diseases, as well as directly impact ecosystem dynamics (Altizer et al. 2011). For example, rinderpest transmitted from livestock to wild mammals in the Serengeti has had catastrophic eects for migrating populations of several ungulates, which in 521

turn has aected vegetation dynamics as grazing pressure has changed (Holdo et al. 2011). Furthermore, birds can actively move both vectors and their parasites; for instance, migratory passerine birds captured in Sweden transported ticks infected with Rickettsia spp., which are pathogens with relevance for human health (Elfving et al. 2010). The infection status of individual birds through time is of relevance for the correct parameterization of epidemiological models; repeated measures eliminate eects due to variation among individuals and facilitate the identication of ecological patterns. In the case of migrants, information derived from recaptured breeding (i.e. nontransient) birds will aid to identify the eects of parasites on dierent life-history traits such as arrival date, territory selection, breeding success and moult. Unfortunately, data from recaptured birds is rarely available from wild populations (Hasselquist et al. 2007, Asghar et al. 2011, Knowles et al. 2011) and most existing data are derived from experimental infections under laboratory conditions (Fallis and Wood 1957, Valkinas et al. 2006). On the one hand, studies on blue tits Cyanistes caeruleus have shown that infection status and parasitaemia are dynamic and can change within a season and across years (Lachish et al. 2011), and that such changes vary for dierent parasite species and individual birds, even at small spatial scales (Knowles et al. 2011, see also Asghar et al. 2011 for great reed warblers Acrocephalus arundinaceus). On the other hand, experiments suggests that in canaries Plasmodium spp. infections can persist for up to eight years and the blood of such birds can be infectious to clean birds for about the same time period (Bishop et al. 1938), even when parasites (i.e. gametocytes) are not detectable in the hosts blood using microscopy (Manwell 1934). Consequently, it is believed that once a bird acquires a haemosporidian parasite, it will remain infected for life in a chronic phase, with relapses during the breeding season, particularly for temperate seasonal habitats (Bennett and Bishop 1990, Valkinas 2005). Parasitaemia can change as the breeding season progresses; early arriving birds can have lower parasitaemia because they are in better condition and have a strong immunological response (Mller et al. 2004), or because the physiological changes during the breeding season (i.e. hormonal changes) that compromise the immune function of the host are still not fully activated (Desser et al. 1968). As the breeding season progresses, males are engaged in competition for resources and mates, energy used in these activities compromises the immune function allowing infections to shed more parasites into the blood stream, particularly for late arriving birds with lower body condition (Rtti et al. 1993). Moreover, parasitaemia can change in dierent years because the condition of birds and the abundance of vectors transmitting parasites are most likely not constant through time. Here, we analyzed Haemoproteus parabelopolskyi infections of blackcaps returning to their breeding grounds in southwestern Germany. These parasites seem to have no signicant eect on the condition of this bird species, other than a short-term weight loss (Valkinas et al. 2006). Hence, we hypothesize that Haemoproteus infection status

and parasitaemia in blackcaps will not be related to arrival time to breeding grounds. Moreover, we hypothesize that infection status of recaptured birds will be constant across years because according to experimental results once a bird is infected it remains so for life in a chronic phase. However, we expect parasitaemia to vary at dierent times because parasite load depends on internal factors of the host (e.g. immune response, Knowles et al. 2011). We used a fragment of the mtDNA cyt b gene and microscopy to determine the infection status and infection intensity of H. parabelopolskyi in recaptured blackcaps. We aimed to answer the following questions: 1) is Haemoproteus infection status and parasitaemia related to the arrival date of blackcaps to their breeding grounds? 2) Do Haemoproteus infection status and parasitaemia of recaptured blackcaps change temporally?

Material and methods

Study species The blackcap is a very abundant passerine across Europe (Prez-Tris et al. 2004). Blackcap populations span the entire range from sedentary to partially and entirely migratory populations that dier in migration distance and orientation (Prez-Tris et al. 2004, Rolshausen et al. 2009). Haemosporidian parasites infecting this bird have been well sampled across western and central Europe (Prez-Tris et al. 2007, Santiago-Alarcon et al. 2011). It is common to nd high prevalence rates ( 70%) of Haemoproteus spp. in blackcaps across their distributional range (Prez-Tris and Bensch 2005a, b, Prez-Tris et al. 2007, Krianauskien et al. 2010, Santiago-Alarcon et al. 2011). In our study site, infections are mostly produced by H. parabelopolskyi (lineages SYAT01 and SYAT02) and prevalence is 70% (Santiago-Alarcon et al. 2011). We studied a population of blackcaps that breed sympatrically in southwestern Germany, but due to a migratory polymorphism overwinters in two distinct areas. A substantial part of the population winters in western Mediterranean areas, namely the Iberian Peninsula and northern Africa (southwest migration, SW). A smaller proportion of the population winters in Great Britain (northwest migration, NW); this migratory polymorphism developed about 60 years ago (Berthold et al. 1992, Bearhop et al. 2005). Blackcaps arriving from distinct winter quarters dier in their arrival times. Blackcaps wintering in Great Britain arrive on average ten days earlier in our population (Rolshausen et al. 2010). Also they choose slightly distinct breeding habitats (Hermes et al. unpubl.). As such, there is temporal and spatial variability as birds coming from Great Britain form mating pairs and occupy their territories rst, enhancing a certain degree of assortative mating (Bearhop et al. 2005, Rolshausen et al. 2010). The development of the migratory polymorphism of this sympatric breeding population has already translated into morphological and genetic dierences between blackcaps with dierent migratory orientation (Rolshausen et al. 2009).

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Field methods We captured blackcaps in southwestern Germany (Freiburg 4800N, 0751E) from 2007 to 2012 by using mist nets aided by tape recordings of their song as a decoy. Each morning, the area was monitored for blackcap activity. This enabled us both to detect and catch newly arrived individuals on our 50 ha study area. We analyzed haemosporidian parasites of 297 blackcaps captured across years (see all blackcaps in statistical analyses), and separately of 27 recaptured birds. Each individual bird was marked with a standard aluminum ring, sexed, and aged based on plumage and skull pneumatization characteristics (Shirihai et al. 2001). We took 75 l of blood from the brachial vein of each captured blackcap using heparinized capillary tubes and stored them at 20C. We prepared two thin blood smears for each bird, they were air dried, xed in 100% methanol and stained with Giemsa in the laboratory. Laboratory methods DNA was extracted using the DNeasy Blood and Tissue Kit. DNA quality was checked on a 1.2% agarose gel. We used parasite genus-specic primers in a nested PCR method (Waldenstrm et al. 2004) to check for avian haemosporidian infection status and to amplify 479 bp of the mtDNA cyt b gene from parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon. For the rst or outer PCR reaction we used a total volume of 10 l per sample. Each reaction contained 1 l of 10 Top Taq Buer, 6.8 l ddH2O, 0.2 l of 400 M dNTPs, 0.5 l from each 10 mM primer, 0.05 l from Top Taq polymerase enzyme and 1 l DNA template. The second or inner PCR reaction had a total volume of 20 l per sample and each reactive was doubled compared to the rst PCR; we used 2 l from the rst PCR as template for the inner reaction. Thermocycler prole for PCR I was 1) 3 min of initial denaturation at 95C, 2) 20 cycles of 30 s denaturation at 95C, 30 s annealing at 50C, 45 s elongation at 72C, and 3) a 10 min nal extension at 72C. The second PCR had a similar thermal prole with a change in the annealing temperature to 57C and 35 cycles instead of 20. We ran 5 l from the second PCR in a 1.2% agarose gel to check for amplication. Positive samples were cleaned with the MinElute Kit, placed in a 96 well plate and sent for sequencing of both forward and reverse strands. Sequences were 479 bp long after editing. Sequences were edited with 4Peaks ver. 1.7.2 ( www.mekentosj.com ). Parasite haplotypes were checked against DNA sequences available in GenBank by using the BLAST algorithm from the NCBI database and also against parasite sequences available at the MalAvi database (Bensch et al. 2009). No new haplotypes are reported in this paper. Determination of infection status of an individual bird using blood samples can be performed using blood smears (microscopy) and a PCR protocol, both of which provide similar prevalence estimates (Valkinas et al. 2008), but under some conditions PCR can be more sensitive than microscopy (e.g. low quality blood smears, Valkinas et al. 2008). Thus, a combination of both microscopy and PCR is recommended to determine the correct infection status of

individual birds (Valkinas et al. 2008). Importantly, both methods are sensitive to infection intensity (Bentz et al. 2006, Knowles et al. 2011). The lower the parasitaemia, the less power both microscopy (Manwell 1934) and PCR have to detect the infection (Knowles et al. 2011). Therefore, in addition to using both microscopy and PCR, it is recommended to check more than one blood smear per bird and to perform PCR replicates of the same sample or from a newly extracted sample of the same bird to increase method sensitivity (Knowles et al. 2011). Based on this, we ran PCRs in triplicate for each bird and we thoroughly checked two blood smears per bird. For microscopy, each blood smear was checked initially for 25 min at low magnication (400) to determine infection status and parasites other than haemosporidian (Valkinas 2005). We then checked 100 elds at high magnication (1000) to determine relative intensity (Valkinas 2005), and we checked from 4000 (for birds with high relative intensity) to 8000 (for birds with low relative intensity) red blood cells to estimate parasitaemia (Godfrey et al. 1987). Mixed infections were detected by both PCR (Prez-Tris and Bensch 2005a) for dierent parasite lineages and microscopy for dierent parasite species and genera. In particular, microscopy was useful at detecting mixed infections of haemosporidians and Trypanosoma spp. parasites. Statistical analyses We divided our analyses in two parts: 1) we analyze a large, multi-year data set containing 297 blackcaps that were captured within the rst 29 d of arrival to breeding grounds to investigate the association between infection status and arrival date, year, and body condition; and 2) we conducted the same analyses on a smaller data set that included only recaptured blackcaps (captured within the rst 31 d of arrival). Because we monitored the study area on a daily basis from mid-March on, we considered the day an individual was rst captured or seen as a proxy for its arrival date. We calculated a day score for each individual, where we set the day the rst blackcap was caught in a given year as day 1. First arrival varied between 17 and 26 of March among years.
All blackcaps

For this analysis infection status was determined only with PCR, as there are several years (20072009) in which blood smears were not prepared. We used general linear models (GLM) to analyze the relationship between day of arrival and infection status. The model was performed using a quasipoisson family distribution We controlled for year eects and body condition eects on infection status. For body condition we used the scaled mass index (Peig and Green 2009, 2010), which is superior to any other condition index developed to date because it takes into account the allometric scaling relationship between morphological measurements (Peig and Green 2010). The scaled mass index was calculated as described in Peig and Green (2009): we used body weight (g) as the mass measurement (Mi) and wing chord (mm) as length measurement (Li). We selected wing chord as length variable 523

because it was best correlated with body weight in comparison with tarsus and culmen. We used the arithmetic mean of wing chord as a standardization index (Lo) to adjust each individual wing length measurement (Li). We conducted standardized major axis (SMA) regression using the natural logarithm of body weight and wing chord. The slope of the best t line of the SMA is the scaling allometric exponent bSMA used to calculate the scaled mass index for each bird. Our bSMA 3.38.
Recaptured blackcaps

Table 1. Results from the all blackcaps data set general linear model (GLM) of infection status in relation to arrival date (day score) to breeding grounds, to body condition index, and to capture year of blackcaps Sylvia atricapilla in southwestern Germany. We implemented a dispersion parameter correction in the model by using a quasipoisson distribution. CI scaled mass index (Peig and Green 2009). GLM (Infection status) Variable Estimate
3

SE
3

p 0.21 0.36 0.21 0.23 0.36 0.29 0.23 0.29

We did not have blood smears for 7 of the 27 recaptured blackcaps and four birds were recaptured at dierent times within the same breeding season, so we did not use them for some analyses and our sample size varied. We rst used a restricted data set, which included birds capture until the 31 d (prevalence) and the 23 d (parasitaemia) after the rst blackcap was capture each year; with this data set we analyzed the relationship between arrival date and infection status (n 42, sample size includes birds for which we had both PCR and microscopy data each time they were captured, sample size refers to repeated measures), and between arrival date and parasitaemia (n 27, sample size includes birds for which we had microscopy information each time they were captured, sample size refers to repeated measures). To analyze infection status we used logistic regression. Because there was overdispersion in the data we corrected the model by using a quasibinomial family distribution. We used general linear models to analyze the relationship between day of arrival and infection intensity (i.e. parasitaemia). Because the variance for parasitaemia was larger for late arriving compared to early arriving blackcaps, we tted our model with a gamma family distribution. We additionally conducted the same two analyses by using an extended data set (included birds that were recaptured up to day score 172) in order to identify changes in parasitism as the breeding season progresses (infection status n 57 [some blackcaps were recaptured three times in dierent years, increasing sample size], parasitaemia n 42). Our recapture data set was to small to include additional factors (i.e. year and condition index) in our GLM analyses. To evaluate if infection status remained constant across years for recaptured blackcaps, we used a McNemars test (Zar 1999). To determine if parasitaemia remained constant through time for recaptured birds, we used a Wilcoxon paired-test with continuity correction (Zar 1999). All analyses were performed in R ver. 2.15.1 ( www.Rproject.org ).

Intercept 3.292 10 Day score 153.5 Year 1.63 CI 178.9 0.076 Day score Year 10.1 Day score CI 0.089 Year CI Day score Year CI 0.005 Null deviance: 216.86 on 296 DF

1.25 2.623 10 170.2 0.9 1.31 1.25 148.4 1.2 847.4 0.9 9.64 1.04 0.073 1.2 0.004 1.04 Residual deviance: 197.46 on 289 DF

is no detectable eect of body condition on the probability of infection by haemosporidians; birds with better condition index have the same probability of infection compared to those in a poorer condition. Moreover, infection probability is not related to the interaction between body condition and arrival time, suggesting that birds with better condition index are not likely to arrive earlier to breeding grounds no matter their infection status. Recaptured blackcaps Haemoproteus infected blackcaps were mostly infected with lineages SYAT01 and SYAT02 (Table 2), corresponding to the morphological species H. parabelopolskyi, which was conrmed with microscopy. One bird infected with H. parabelopolskyi in 2010, changed its infection in 2011 with Trypanosoma (Table 2). One bird had a double infection with Haemoproteus and Trypanosoma the two times it was captured, in 2010 and in 2011 (Table 2). Infection results from PCR and microscopy were in general agreement, but there were ve microscopy positive samples misclassied by PCR as negative (Table 2). During the rst 31 capture days (and utilizing the restricted data set), we found a marginally signicant eect of H. parabelopolsky infection on arrival time of blackcaps to breeding grounds (Table 3, Fig. 1), indicating that uninfected birds are marginally more likely to reach breeding grounds earlier compared to infected individuals. Using an extended data set, up to day score 172, we detected a higher probability of infection in blackcaps that were recaptured later in the season (Table 4, Fig. 2). Infected birds had marginally signicant lower parasitaemia if captured earlier in the season compared to those captured later in the breeding season (Table 4). However, the relationship between capture date and parasitaemia was triangular, meaning that birds with low infection intensity were not necessarily captured earlier in the breeding season compared to those with high parasite loads (Fig. 3). This pattern holds when removing outliers, which in some cases reduces the time span (capture date) of data sets (Fig. 3). Removing outliers is not warranted when

Results
All blackcaps The eect of Haemoproteus parabelopolsky infection on arrival time of blackcaps to breeding grounds was found non signicant (Table 1), indicating that infected and uninfected birds do not dier in their arrival time to breeding grounds. This result holds after controlling for year eects and for body condition of individual birds (Table 1). There

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Table 2. Infection status, relative intensity, parasitaemia and arrival day (day score) to breeding grounds in southern Germany from recaptured blackcaps Sylvia atricapilla sampled between years 2007 and 2012. There were no blood smears for 7 of the 27 recaptured blackcaps, and four birds were recaptured at different times within the same season. Ring numbers highlighted in black indicate that birds were recaptured in the same year but at different season. For bird C2T9558 the rst and second data for each category are from different seasons in the same year (2010) and its third datum for each category is from a recapture in a different year (2012). Underlined ring numbers refer to samples that were positive with microscopy, but negative with PCR. H Haemoproteus, L Leucocytozoon, positive, negative, information not available because no such sample existed, positive, negative, l icroscopy, but negative with PCR.ive by microscopy and negative by PCR.2011 paper, the correct it was not possible to obtain a good sequence, so haplotype is unknown.

Infection status PCR Haplotype 1st - (Infected with Trypanosoma) 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 0.275 0.225 0.225 0.05

Infection status microscopy

Relative intensity nas (codes in Valkiu 2005)

Parasitaemia (%) 2nd 3rd 0.025 0.05 0.0125 1st

Day score 2nd 3rd 17 99 4 6 15 9 1 7 7 7 11 12

Ring number

1st

2nd

3rd

C2L6233 C2T9534 C2L6162 C2L6168 C2L6222 C2L6179 H-SYAT01 H-SYAT01

H-SYAT01

H-SYAT02 H-SYAT01

H-SYAT02 H-SYAT01 L-SFC8 H-SYAT01 H-SYAT02 H-SYAT01 H-SYAT02 H-SYAT01

C2T9517 C2L6252 C2T9544 C2T9634 C2H9502 C2T9653 C2T9561 C1E2612 C2T9659 C2T9558 C2T9623 C2T9512 C2L6242 C1E2661 C1E2713 C1L6320 C2L6187 C1V0680 C1V0654 C2G4322 CT41185

H-SYAT02

H-SYAT01&02 NS H-SYAT01 & L-IBT2 H-SYAT (several lineages)

H-SYAT01 H-SYAT01 H-SYAT02 H-SYAT01 H-SYAT02 H-SYAT01

H-SYAT01 H-SYAT02 H-SYAT01

0.1 0.075 0.075 0.075 1.57 0.075 0.175 1.07

0.025 0.325 0.05 0.0125 0.7 0.025

0.075 0.0125 0.0125

80 18 100 140 25 141 130 172 141 130 140 65 17 3 12 67 10 24 19 31 22

13 15 15 15 18 21 23 51 56 141 172 95 80 10 19 9 12 8 11 17 11

19 22 13

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Table 3. Results from the restricted data set general linear models (GLMs) of infection status and parasitaemia in relation to arrival date (day score) to breeding grounds in southwestern Germany of recaptured blackcaps Sylvia atricapilla. We implemented a dispersion parameter correction in the logistic regression model (infection status) by using a quasibinomial distribution. Parasitaemia was square-root transformed and we used a GLM with a gamma family distribution to improve model t. rbc red blood cells. Logistic regression (infection status) Variable Estimate SE 0.816 0.053 t p Variable Intercept 1.32 Day score 0.094 Null deviance: 58.22 on 41 DF 0.1142 1.61 1.75 0.086 Residual deviance: 54.62 on 40 DF GLM (parasitaemia) Estimate SE 0.010 0.0054 t p Intercept 0.0821 Infected rbc 0.0038 Null deviance: 7.88 on 26 DF 5.02 0.001 0.492 0.69 Residual deviance: 7.79 on 25 DF

analyzing parasite data, however, because parasites normally exhibit aggregated distributions in host populations, conforming to a negative binomial distribution (Wilson and Grenfell 1997), which means that few individuals in the population will have heavy parasite loads and most will have few parasites or be uninfected (Wilson et al. 1996, Wilson and Grenfell 1997). McNemars test was not signicant (2 0.363, p 0.5), indicating that infection status remained constant through time for individual birds; however, some birds changed their infection status between seasons within the same year or in dierent years (Table 2). Parasitaemia was not constant through time (V 159, p 0.01); it varied among seasons and among years for some birds (Table 2). Parasite load was higher later in the season (summer) and its variation across years also depended on the season at which the bird was captured (Table 2).

Discussion
We studied the relationship between parasitism and arrival date in blackcaps using information on birds sampled from
1

0 0 5 10 15 20 Day score 25 30

Figure 1. Logistic regression showing the relationship between infection status and arrival date (day score) to breeding grounds of the restricted data set of recaptured blackcaps Sylvia atricapilla (n 42, latest day score 31). The line is the best-t model to the data using the logistic model 1/[1 ey].

2007 to 2012 in a population from southwestern Germany. We found that in this blackcap population infection by haemosporidian parasites has no relationship to arrival date of blackcaps, but infection by Haemoproteus parabelopolskyi has a marginally signicant eect on arrival date of recaptured blackcaps, somewhat delaying their arrival to breeding grounds. The result for the blackcap population is in agreement with our initial hypothesis, where no delay in arrival time was expected given that haemosporidians (in particular, H. parabelopolskyi) have mild tness consequences on blackcaps (Valkinas et al. 2006). Results restricted to recaptured blackcaps are not entirely consistent with our initial hypothesis. One explanation for the marginal delay can be that less t chronically infected birds or birds that acquire an infection during migration (e.g. at stopover sites) are more challenged to accumulate energy reserves for migration, using energy for physical maintenance instead of fuel deposition (Metzger and Bairlein 2011), which delays their departure time (DeGroote and Rodewald 2010). This is unlikely, however, given that there are no dierences in mass deposition rate between haemosporidian infected and uninfected blackcaps (Arizaga et al. 2009), and we also detected no dierences in body condition between infected and uninfected birds (Table 1). In addition, the temporary weight loss they experience at the peak (acute phase) of Haemoproteus infection (Valkinas et al. 2006) is unlikely to persist during (and thereby aect) spring migration as prevalence is already very high (85%) in juvenile blackcaps before their autumn migration (Santiago-Alarcon et al. 2011). During spring migration and arrival to breeding grounds (mid-March to early April) some infections can still be chronic, in which case we did not detect them because we used PCR and microscopy on peripheral blood. Taken together, we found no evidence of haemosporidians aecting spring migration in blackcaps over consecutive years. This is consistent with the relative weak eects of H. parabelopolskyi found on blackcaps earlier (Valkinas et al. 2006). We found that individuals captured earlier in the season are more likely to be uninfected or if infected have lower parasitaemia compared to the same individuals captured later in the season. There are three proximate mechanisms which can explain this result. Blackcaps are more likely to get infected later in the season because parasite abundance increases in early summer and local transmission occurs on the breeding grounds (Santiago-Alarcon et al. 2011) and because the richness and abundance of competent vectors increases as well (Santiago-Alarcon et al.

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Infection probability

Table 4. Results from the extended data set general linear models (GLMs) of infection status and parasitaemia in relation to capture date of blackcaps as the breeding season progresses (i.e. temporal variation). We implemented a dispersion parameter correction in the logistic regression model (infection status) by using a quasibinomial distribution. Parasitaemia was square-root transformed and we used a GLM with a gamma family distribution to improve model t. rbc red blood cells. Logistic regression (infection status) Variable Estimate SE 0.393 0.010 t p Variable Intercept 0.293 Day score 0.0213 Null deviance: 76.8 on 56 DF 0.458 0.746 2.080 0.042 Residual deviance: 69 on 55 DF GLM (parasitaemia) Estimate SE 0.0041 0.00006 t p Intercept 0.022 Infected rbc 0.00012 Null deviance: 55.41 on 41 DF 5.52 0.001 0.076 1.82 Residual deviance: 53.01 on 40 DF

2013). Finally, birds often have a suppressed immune system during the reproductive season owing to hormonal changes that could inuence changes from the chronic to the acute phase and then increase chances of a new infection (Valkinas 2005). As the breeding season progresses, energy used in reproductive activities (e.g. egg production, ospring provisioning) compromises the immune function allowing infections to shed more parasites into the blood stream (acute phase of infection), particularly for late breeding birds that are in poorer condition (Rtti et al. 1993, Mller et al. 2004). In particular, a temporary weight loss induced by the acute phase of haemsporidian infection in blackcaps (Valkinas et al. 2006) could inuence the outcome of competitive encounters such as ghts for territories. Accordingly, uninfected birds should have a competitive advantage over infected individuals, and among infected individuals those better able to control parasite load will be expected to be more successful at acquiring better breeding territories (i.e. more food and less exposure to parasites and vectors) and higher quality mates. For example, ectoparasites such as chewing lice can delay arrival to breeding grounds in male
1

barn swallows Hirundo rustica, which favors uninfected birds during intraspecic competition for high quality territories and mates in breeding grounds (Mller et al. 2004). Moreover, male barn swallows with a higher T-cell mediated immune response arrive earlier compared to males with a weaker response (Mller et al. 2004). Therefore, we cannot rule out that parasitism by Haemoproteus in blackcaps has an indirect tness eect that is relevant for what happens throughout the reproductive stage as a consequence of a late breeding start, which is likely to negatively aect territory establishment, mate acquisition, and raising successful broods. Phylogenetic analyses reveal only two haemosporidian parasite exchanges between African and European resident birds via migrants, as well as few parasite lineages common to both migratory and resident birds (Hellgren et al. 2007). This indicates that in the European-African migration route introduction of parasites to resident faunas is rare and that parasite expansion from one biogeographical zone to another happens slowly through evolutionary time, which varies depending on parasite genus (expansion is faster for Plasmodium lineages; Hellgren et al. 2007). Despite this slow haemosporidian dynamic between EuropeanAfrican quarters, just one successful parasite exchange such as Plasmodium lineage SGS1 can impact the ecology of
120 Parasitaemia (# parasites / 8000 rbc)

Infection probability

100 80 60 40 20 0 0 50 100 Day score 150

0 0 50 100 Day score 150

Figure 2. Logistic regression using the extended data set of recaptured blackcaps (n 57, latest day score 172) showing the relationship between infection status and capture date of recaptured blackcaps Sylvia atricapilla throughout the breeding season. The line is the best-t model to the data using the logistic model 1/[1 ey].

Figure 3. Relationship between parasitaemia and capture date (extended data set) of recaptured blackcaps Sylvia atricapilla throughout the breeding season (note the triangular shape).

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resident and migrant species through its health eects (Palinauskas et al. 2008, 2011). In general, novel emergent diseases seem to result from rare dispersal events or host switches (Ewald 1994), and this is true for other migration systems as well (e.g. rinderpest in Serengeti ungulates, Holdo et al. 2011). Hence, rare parasite expansion events can have deep ecological consequences for avian hosts. In the case of blackcaps, the morphospecies H. parabelopolskyi is highly prevalent and the most common haemosporidian parasite found across European blackcap populations (Prez-Tris and Bensch 2005a, b, Prez-Tris et al. 2007, Krianauskien et al. 2010, Santiago-Alarcon et al. 2011). Lineage SYAT01 from this morphospecies is found both in resident African and European bird populations, having probably originated from European bird populations (Fig. 2 in Hellgren et al. 2007). In this study, we have identied H. parabelopolskyi (lineages SYAT01 and SYAT02) as the most common parasite infecting blackcaps, and as the only Haemoproteus parasite infecting recaptured blackcaps; this parasite has been found infecting juvenile birds, indicating that is locally transmitted (Santiago-Alarcon et al. 2011). Hence, we suggest that H. parabelopolskyi is largely responsible for the observed marginal eect on recaptured birds (i.e. uninfected birds are captured earlier in the season compared to infected ones). We have also detected mixed infections of dierent haemosporidian genetic lineages (Santiago-Alarcon et al. 2011, Table 2) and also of haemosporidians with Trypanosoma in the present and other studies (Table 2, Valkinas et al. 2004). Mixed infections might have typically stronger tness consequences for birds compared to single infections (Evans and Otter 1998, Davindar and Morton 2006, Palinauskas et al. 2011); however, Marzal et al. (2008) have found positive tness eects of mixed infections (i.e. birds with double infections had a higher reproductive success). Thus, the general eects of mixed infections are not well resolved (Marzal et al. 2008). Our results showed that infection status is constant through time, indicating that infected birds are unlikely to lose their infection, at least in the time period considered here. This result is in agreement with the observation that once a bird is infected with a haemosporidian parasite, it will likely remain infected for life (Bishop et al. 1938, Manwell 1934, Bennett and Bishop 1990, Valkinas 2005). However, some birds changed their infection status either from positive to negative (e.g. blackcap C2T9534, Table 2) or from negative to positive (e.g. blackcap C2L6168, Table 2); this change might represent chronic infections, in which case we were not able to detect them (i.e. false negatives) because we analyzed parasites in peripheral blood and not in internal organs where parasites are found prior to relapses during the chronic phase of infection. Despite this methodological constraint, for practical purposes, apparent recovery of individuals (i.e. parasites are not present in blood, but have receded to internal organs) can be taken as a full recovery in epidemiological terms because the infectious parasite stage (gametocytes) is not available for vector transmission. Therefore, the relevant epidemiological issue for parasites transmitted by blood feeding vectors is to determine if the parasite is circulating in the hosts peripheral blood or not, and both 528

of our methods were in general agreement (see also Valkinas et al. 2008, Asghar et al. 2011). As predicted and opposite to prevalence, parasitaemia was not constant through time (but see Asghar et al. 2011). This dierence can be due to the fact that prevalence and parasitaemia are dierent parameters of an infection (Knowles et al. 2011, Lachish et al. 2011). Whereas prevalence or transmissibility of a parasite is inuenced mostly by external factors (e.g. temperature aecting availability of vectors), the number of parasites infecting a host is mostly determined by internal factors (e.g. immune response). Birds are not expected to have constant health or tness through time; this depends on resources and stochastic factors. Hence, in good years infected birds are expected to control parasite numbers better compared to years where food resources are scarce. Furthermore, temporal dynamics of parasitaemia seem to depend on parasite species and genetic lineage. For instance, parasite loads of lineages GRW1 and GRW2 infecting great read warblers A. arundinaceus are highly repeatable for individual birds across years, but it is not for lineage GRW4 (Asghar et al. 2011). Hence, parasitaemia will vary across individual birds, among dierent seasons, and across years due to dierences in immune response, to environmental conditions aecting the physical state of individual birds (e.g. food abundance), and to the implicated parasite species.
Acknowledgements We thank Jana Bhler, Carlo Catoni, Marie Lucas, Marie Melchior, Kalliope Stournaras, Sebastian Vetter, Frederick Wehrle, Rebecca Bloch, Claudia Hermes and Gregor Rolshausen for assistance during eldwork. Comments from S. Bensch greatly improved the quality of the paper. This work was supported by the Deutsche Forschungsgemeinschaft (HMS, grant number 1008/6-1), and by the Alexander von Humboldt Foundation (DSA, post-doctoral grant). Data accessibility We provide an excel le (Supplementary material Appendix A1) that contains the data used to perform all presented analyses. The data le will be deposited in Dryad.

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Supplementary material (Appendix JAV-00181 at www. oikosoce.lu.se/appendix ). Appendix A1.

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