Urban Ecosyst DOI 10.1007/s11252-015-0494-0

Avian haemosporidian parasites in an urban forest and their relationship to bird size and abundance Diego Santiago-Alarcon 1,3 & Ian MacGregor-Fors 2 & Katharina Kühnert 3 & Gernot Segelbacher 4 & H. Martin Schaefer 3

# Springer Science+Business Media New York 2015

Abstract Urbanization has been identified as a threat to biodiversity due to landscape modifications. Studies of parasite ecology in urbanized areas lagged behind those made on macro organisms. Here we studied infection prevalence of haemosporidian parasites in an avian community of an urban forest from Germany, and its relationship with bird abundance and body mass. We used PCR to amplify a fragment of the mtDNA cyt b gene to determine the infection status of birds, and bird point counts to determine bird relative abundances. The avifauna was dominated by two small sized insectivore passerines (Parus major, Cyanistes caeruleus), representing ~40 % of the total bird records. The highest haemosporidian prevalence was recorded for Turdus philomelos (100 %) and for Fringilla coelebs (75 %). Bird abundance and body mass were positively associated with infection status for two haemosporidian genera: Plasmodium and Leucocytozoon. Infection rate was lower in juveniles compared to adult birds. We recorded a total of 7 Plasmodium, 26 Haemoproteus, and 10 Leucocytozoon lineages. Avian malaria (P. relictum) was detected infecting 5 individuals of P. major, the most abundant species in the community. These results, together with those of previous studies at the same site, suggest that potentially any of the genetic haemosporidian lineages detected in this urban forest can be transmitted across native and pet bird species, and to species of conservation concern housed at aviaries.

* Diego Santiago-Alarcon [email protected] 1

Red de Biología y Conservación de Vertebrados, Laboratorio de Ecología de Vertebrados e Interacciones Parasitarias, Instituto de Ecología A.C., Xalapa 91070 Veracruz, Mexico

2

Red de Ambiente y Sustentabilidad, Laboratorio de Ecología en Ambientes Perturbados, Instituto de Ecología A.C., Xalapa 91070 Veracruz, Mexico

3

Department of Ecology and Evolutionary Biology, University of Freiburg, Freiburg, Baden-Württemberg, Germany

4

Department of Wildlife Ecology and Management, University of Freiburg, Freiburg, Baden-Württemberg, Germany

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Keywords Urban parasitology . Zoonosis . Plasmodium . Haemoproteus . Leucocytozoon . Avian malaria

Introduction Urbanization has been identified as a threat to biodiversity (Czech et al. 2000; McKinney 2002; Berkowitz et al. 2003; Alberti 2008; Shochat et al. 2010). Parasitological studies in urbanized areas to date are lagging behind those conducted on macro organisms (Delgado-V and French 2012; Santiago-Alarcon et al. 2012a, 2013a). This limits our understanding of the ecological patterns and processes of parasites in cities and adjacent areas (Delgado-V and French 2012). Urbanization affects the ecology of wildlife diseases directly and indirectly (Bradley and Altizer 2007). For example, by increasing species diversity it is possible to create dilution effects (Keesing and Ostfeld 2012), but amplification effects are also possible through increases in species diversity, in particular if the density and abundance of key host species adapted to urban settings augment (Bradley and Altizer 2007; Randolph and Dobson 2012). Urbanization modifies among-species contact rates and parasite distribution, for instance by altering vector-feeding preferences (e.g. West Nile virus: Kilpatrick et al. 2006; avian malaria: Santiago-Alarcon et al. 2012a, 2013a). Birds are among the best-studied groups in reference to their parasites and infectious diseases (Thomas et al. 2007; Atkinson et al. 2008), but little is known on the ecological and evolutionary dynamics of bird-parasite interactions in urban and sub-urban areas (Delgado-V and French 2012). Most studies that report the parasite species list infecting birds inhabiting cities around the world are restricted to a few urban adapted species (e.g. pigeons [Columba livia], house sparrows [Passer domesticus], house finches [Haemorhous mexicanus], starlings [Sturnus vulgaris], blackbirds [Turdus merula], corvids [Corvus spp.]), and it is for those species that we also have the best information regarding parasite health effects (Delgado-V and French 2012). Some bird species will experience a larger infection rate in urban areas whereas others will lose their parasites or have no difference at all when comparing urban and rural sites (e.g. Fokidis et al. 2008; Martin and Boruta 2014). Specific host attributes such as body size and age, and ecological traits like abundance can determine the relevance of a species in a parasite life cycle and its role as potential reservoir. Given that some bird species can attain large abundances, survive better, and reach larger sizes in urban areas, it is important to understand how such attributes relate to infection rates (Martin and Boruta 2014). In a large survey of European birds, Scheuerlein and Ricklefs (2004) identified a strong positive correlation between haemosporidian infection prevalence and body size; they also found a positive correlation with age and the length of the period between hatching and fledging. Hence, the better survival of some birds in urban areas and their increased abundance (Shochat 2004; Evans et al. 2009) could make them an important reservoir for pathogens (e.g. Eurasian blackcap [Sylvia atricapilla], Santiago-Alarcon et al. 2011). Avian haemosporidians are vector-borne intracellular parasites from the genera Plasmodium, Fallisia, Haemoproteus, and Leucocytozoon that can have negative fitness effects on their hosts, such as high mortality in endemic insular birds (e.g. Atkinson et al. 2000; Yorinks and Atkinson 2000), lower reproductive output, lower body condition, and hypertrophy of internal organs in several passerine birds (e.g. Merino et al. 2000; Marzal et al. 2005; Palinauskas et al. 2008). Although parasitologists long believed that avian haemosporidian parasites were host specific (Bennett et al. 1993, 1994), recent studies have shown that

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avian haemosporidian parasites have an evolutionary history of host switching with little codivergence (Bensch et al. 2000; Ricklefs et al. 2004; Santiago-Alarcon et al. 2014). Haemosporidians are mainly transmitted by five families of Diptera: Culicidae, Hippoboscidae, Simuliidae, Ceratopogonidae and Psychodidae (Valkiūnas 2005; SantiagoAlarcon et al. 2012b). Until recently it was believed that most vector species had specialized feeding habits. However, recent molecular data show that some vector species have generalized feeding habits in urban areas (Jansen et al. 2009; Santiago-Alarcon et al. 2012a, b, 2013a). Host generalists are of special interest because they are capable of feeding on different vertebrate groups, and thus can facilitate the emergence of new diseases by spreading parasites to new hosts (e.g. Santiago-Alarcon et al. 2012a, 2013a). Hence, because urbanization can alter vector-feeding preferences, it is a priority to study the biodiversity of parasites infecting wild vertebrates inhabiting urban areas as this can have incidence on other species of economic (e.g. poultry, Johnson et al. 1938; Kissam et al. 1975), conservation (e.g. endemic birds, Levin et al. 2009; birds kept at zoos for reintroduction programs, Carlson et al. 2011; Pacheco et al. 2011), and medical importance (e.g. viral diseases affecting humans, Kilpatrick et al. 2006). In this work, we studied infection prevalence of haemosporidians in an avian community of an urban forest of the city of Freiburg in Germany. We analyzed the abundance of the bird community and its relationship to haemosporidian prevalence for understory birds captured with mist nets. Considering that many vector species are generalists (Jansen et al. 2009; Santiago-Alarcon et al. 2012a, b, 2013a), and that many avian haemosporidians are able to successfully infect a diverse array of bird hosts (Hellgren et al. 2009), we expected to find a positive relationship between host abundance and prevalence because prevalence is a population parameter that depends mostly on external factors (e.g. temperature, humidity, contact rate between parasites and hosts, e.g. Knowles et al. 2011); hence, as a host is more abundant the probability of encountering a parasite increases. We controlled for body size while analyzing abundance given that body size is negatively associated with abundance and it is expected to correlate positively with infection risk (Scheuerlein and Ricklefs 2004). Because adult birds are expected to have a higher probability of being infected given that they have had a longer period of exposure to parasites (Valkiūnas 2005) and because longevity can be increased in urban areas (Evans et al. 2009), we also investigated the relationship between infection prevalence and age for bird species with sufficient sample size, and expected a larger proportion of adults being infected.

Materials and methods Fieldwork Fieldwork was carried out in the urban forest Mooswald, located in the upper Rhine valley, in the city of Freiburg in Southwestern Germany (48°00′N 07°51′E) between 2nd of June and 14th of July 2011, which is the period when all breeding species are present (i.e. all migratory birds have arrived and not yet departed). This area is used intensively for recreational activities and is characterized by many trails and paths that are frequently used by joggers or walkers. Every 5 to 10 years thinning takes place. The dominating tree is European ash (Fraxinus excelsior), followed by French oak (Quercus robur) and Northern red oak (Quercus rubra). Trees found in this forest have an age of about 80 to 120 years (Markus Müller, pers. com.). As of 2012, the city of Freiburg has an area of 153.06 km2, 224,191 inhabitants, and a density of

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1465 inhabitants/km2 (Statistical Year Book, Germany, Extract Chapter 2: populations, families, and living arrangements. 2012. Federal Statistical Office of Germany: https://www. destatis.de/EN/Publications/Specialized/Population/StatYearbook_Chapter2_5011001129004. pdf?__blob=publicationFile). We used five to ten mist nets to catch understory birds. Nets were placed across the study site, covering an area of 64 ha during the sampling period. Nets had a length of 6, 9, or 12 m and were 2.5 m high. Nets were opened at sunrise and closed at 12 pm. Bird blood was obtained by brachial venipuncture. The maximum volume obtained was 75 μl. Blood samples were frozen immediately after returning from the field at −20 °C. We used 25 m radius point counts to census the summer avifauna from Mooswald forest. Point counts were separated by a distance of 200 m and had duration of 10 min. We recorded all birds sighted or heard within and outside of the 25 m radius (Hutto et al. 1986). We conducted twelve point counts and did five repetitions of each point throughout the summer.

Laboratory methods DNA was extracted using the DNeasy Blood and Tissue® Kit (QIAGEN, Hilden). DNA quality was checked on a 1·2 % agarose gel. We used parasite genus-specific primers in a nested PCR method (Hellgren et al. 2004) to check for avian haemosporidian infection status and to amplify 540-bp of the mtDNA cytochrome b (cyt b) gene from parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon. For the first or outer PCR reaction we used a total volume of 10 μl per sample. Each reaction contained 1 μl of 10°— Top Taq Buffer (QIAGEN, Hilden), 6·8 μl of double-distilled H2O, 0·2 μl of 400 μM dNTPs, 0·5 μl from each 10 mM primer, 0·05 μl from Top Taq polymerase enzyme (QIAGEN, Hilden), and 1 μl of DNA template. The second or inner PCR reaction had a total volume of 20 μl per sample and each reactive was doubled compared to the first PCR; we used 2 μl from the first PCR as template for the inner reaction. Thermocycler profile for PCR I was (1) 3 min of initial denaturation at 95 °C, (2) 20 cycles of 30 s denaturation at 95 °C, 30 s annealing at 50 °C, 45 s elongation at 72 °C, and (3) a 10 min final extension at 72 °C. The second PCR had a similar thermal profile with a change in the annealing temperature to 57 °C and 35 cycles instead of 20. We ran 5 μl from the second PCR in a 1·2 % agarose gel to check for amplification. Positive samples were cleaned with the MinElute® Kit (QIAGEN, Hilden), 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 (http://www.mekentosj.com) and BioEdit® (Hall 1999). 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).

Statistical analyses In order to characterize the bird communities of the studied area, we used rank/abundance (= Whittaker) plots. We used this approach, as these types of plots represent the species abundance distribution of a community graphically, allowing us to describe the diversity of a community, and reflecting the success of the implied species to compete for limited resources and conditions (Magurran 2004). In order to differentiate among abundant and rare species, we used a data-dependent cut-off (k=n/Species observed) recently proposed by Chao and Chiu (2013), which basically represents species average frequency. With the k-value calculated, we

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separated abundant species in two groups, as we found an important drop-off in relative abundances between the recorded tits (i.e., Great tit, Eurasian blue tit) and the rest of the abundant species. Thus, we divided the recorded bird community in three abundance groups: highly abundant (2 species: Great tit [Parus major], Eurasian blue tit [Cyanistes caeruleus]), moderately abundant (6 species: Eurasian blackcap [Sylvia atricapilla], Common blackbird [Turdus merula], Eurasian nuthatch [Sitta europaea], Great spotted woodpecker [Dendrocopos major], European robin [Erithacus rubecula], Eurasian wren [Troglodytes troglodytes]), and rare (17 species: Fig. 1). We used the software Quantitative Parasitology 3.0 (Rózsa et al. 2000) to calculate infection prevalence and its respective 95 % confidence intervals (CI) for each bird species for which we had ≥10 captured individuals or that were ranked as highly or moderately abundant in the observational surveys (see above). Confidence intervals were calculated with the Sterne’s exact method (Reiczigel 2003). We used Fisher’s exact test (Rózsa et al. 2000) to compare differences in prevalence between infections by Leucocytozoon and Plasmodium/ Haemoproteus for bird species with N≥10 (see Table 1), and between adults and juveniles for species that had sufficient sample sizes (i.e. Blackbird, Robin, and Great tit). We conducted generalized linear models (GLM) using a binomial family distribution to analyze the influence of host body mass and abundance on infection probability; we conducted separate GLMs for each parasite genus (i.e. Plasmodium, Haemoproteus, and Leucocytozoon) and for individuals with mix infections. Analyses were conducted in R 2.15.2 (http://www.R-project.org). All GLM analyses were conducted only on seven understory bird species that had an N≥10 (Table 1); of these, only the Eurasian blackcap is a long-distance migrant, all other bird species are resident or undertake short local movements. We have studied the Eurasian blackcap in

Fig. 1 Summer avifauna from Mooswald forest in Freiburg, Germany. Bird species are presented in decreasing order of abundance, and they are categorized in highly abundant, moderately abundant, and rare (see methods). From species categorized as rare only two are named because they represent species that are common and widely distributed in Europe. For exact figures of relative abundance of all species see Table 1

2.8

1.6

1.4

Garrulus glandarius

Certhia familiaris

Turdus philomelos

5

3

Columba palumbus

Oriolus oriolus

6.6

5.6

Phylloscopus collybita

Aegithalos caudatus

9

8

Troglodytes troglodytes

Fringilla coelebs

10.2

Erithacus rubecula

3

15

12.4

Sitta europea

Dendrocopos major

21

19

Turdus merula

6

–

12

12

0

–

3

– –

–

–

0

1

12

0

5

0

0

13

6

0

20

Infected birds (PCR)

1

5

16

10

61

4

18

36.2

20.4

43

Cyanistes caeruleus

41

Parus major

Sample size (N)

Sylvia atricapilla

Relative abundance

Bird species

100

0

–

–

–

0

20

75

0

8.19

0

0

61.9

33.3

0

46.5

Prevalence (%)

–

–

75.7–100

Plasmodium LIN3, P. circumflexum TURDUS1; Haemoproteus SYAT32; Leucocytozoon ChL4,

– –

–

– –

–

–

–

– –

1.03–65.7 –

60

–

–

–

–

–

23.6

9

16.65

– –

– – Plasmodium LINN1; Haemoproteus balmorali H-ROBIN1; Leucocytozoon STUR1 –

84.9

Plasmodium LINN1, and LIN3; Haemoproteus WTTH61H, H. minutus (98 %) H-TURDUS2; Leucocytozoon ChL4

17.9

– See Santiago-Alarcon et al. (2011) for a full list

18.25

Average body massa

Plasmodium LIN3, P. relictum P22, SGS1, and GRW11; Haemoproteus majoris CWT4, WW2, and PARUS1; Leucocytozoon IPARUS4, and AK04

Parasite species and lineages

Haemoproteus CCF1, CCF2, SYAT32, and SYAT35, H. pallidulus lineage SYAT03

50–90.9

0–29.1

3.3–17.8

0–52.7

0–63.1

40.3–80.2

15.6–58.6

0–41.1

32.1–61.7

95 % CI

Table 1 Summer avifauna from Mooswald forest in Freiburg, Germany. Relative abundances were calculated with point counts of 25 m radius (Hutto et al. 1986), we used PCR to detect haemosporidian infection (N refers to the number of birds captured in mist nets from which a blood sample was obtained). Prevalence 95 % confidence intervals (CI) were calculated with the Sterne’s exact method (see Reiczigel 2003). 95 % CI for prevalence are reported for bird species that had an N≥10 or that are ranked as highly or moderately abundant (see Fig. 1). Average body mass (grams) is reported only for birds that had an N≥10. NC = not detected during point counts

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a

3

0

0

100

33.3

– 0

–

–

0

–

100

–

Prevalence (%)

–

Leucocytozoon ANLA3 Unidentified Leucocytozoon –

– – –

– –

–

– –

–

–

– –

–

Unidentified Leucocytozoon

– –

–

STUR1, and ANLA3

Parasite species and lineages

–

95 % CI

–

–

–

– –

–

–

–

–

–

–

Average body massa

Data sources: Glutz (1987), Flegg and Cox (1977), Pikula (1973), Berthold and Querner (1982), and Hand Book of the Birds of the World Alive (HBW Alive: www.hbw.com

NC

Sylvia borin

1

1

3

0.2

NC

Muscicapa striata

Acrocephalus palustris

1

– 0

– 4

0.2 0.2

Carduelis chloris Poecile palustris

– –

0.4

0.2

Picus viridis

Accipiter nisus

–

– 0

–

1

0.6

0.6

Regulus regulus

Dendrocopos medius

–

1

1

0.8

Coccothraustes coccothraustes

–

–

1

Corvus corone

Infected birds (PCR)

Sample size (N)

Relative abundance

Bird species

Table 1 (continued)

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detail in relation to its haemosporidian parasites and migratory strategies elsewhere (SantiagoAlarcon et al. 2011, 2013b); hence, we have not included the migratory vs. non-migratory factor in our statistical analyses.

Results We recorded a total of 27 bird species in our study area and captured a total of 216 birds in mist nets (Table 1). Although our study does not aim to compare this community to others, it is noteworthy that our community is dominated by two small sized insectivore passerines (i.e., Great tit, Eurasian blue tit) with their relative abundances representing ~40 % of the total recorded relative abundances in point counts (Fig. 1). Most moderately abundant species are insectivores; two are considered frugivores (despite the fact that they are insectivores during the breeding season: Eurasian blackcap, Common blackbird; HBW 2013). The rest of the recorded species, classified here as rare (considering a data-dependent cut-off - see methods for details) are mainly insectivores, with some seed-eaters (e.g., Common wood pigeon [Columba palumbus], Hawfinch [Coccothraustes coccothraustes], European greenfinch [Carduelis chloris]), omnivores (e.g., Eurasian jay [Garrulus glandarius], Carrion crow [Corvus corone]), and carnivores (i.e., Eurasian sparrowhawk [Accipiter nisus]) species (HBW 2013). The highest haemosporidian prevalence was recorded for the Song thrush (Turdus philomelos, 100 %) and for the Common chaffinch (Fringilla coelebs, 75 %; see Table 1), both of which are ranked as rare in the bird community (Fig. 1). The Great tit (P. major) had the highest abundance and an infection prevalence of 46.5 %, followed by two moderately abundant species, T. merula and S. atricapilla with 61.9 and 33.3 % infection prevalence respectively. The European robin (E. rubecula) was the bird with the largest sample size (N= 61), it was ranked as moderately abundant but had the lowest prevalence (8.2 %) compared to species with an N≥10 (Table 1). When analyzing prevalence for all parasite genera we detected that both abundance and body mass have a significant positive effect on infection probability (Table 2); the same pattern was observed for the genera Plasmodium, Leucocytozoon, and for birds with mixed infections (Table 2). We found, however, no effect of any variable on infection probability for parasites of the genus Haemoproteus. We also detected an interaction pattern between abundance and body mass on infection probability, these two variables are negatively associated, indicating that there is a trade off between being a small abundant bird species or vice versa on the probability of acquiring an infection (Table 2). We identified a total of 7 Plasmodium, 26 Haemoproteus, and 10 Leucocytozoon lineages (Table 1). Avian malaria (Plasmodium relictum lineages P22, SGS1 and GRW11) was detected infecting 5 individuals of Parus major, the most abundant bird in the community (Fig. 1). From Haemoproteus parasites, H. minutus lineage L-TURDUS2, H. pallidulus lineage SYAT03, H. parabelopolskyi lineages SYAT01 and SYAT02, and Haemoproteus lineages CCF1, CCF2, SYAT32, and SYAT35 commonly infect two moderately abundant species (S. atricapilla and T. merula) and one rare species (F. coelebs) that are widely distributed across Europe (Križanauskienė et al. 2010; Santiago-Alarcon et al. 2011); these Haemoproteus lineages are known to be transmitted by different species of Culicoides (Diptera: Ceratopogonidae) vectors in the sampled community (Santiago-Alarcon et al. 2012a, 2013a). Infection rate of Leucozytozoon parasites was significantly lower in comparison to Haemoproteus/Plasmodium parasite infections for the following species: T. merula (p= 0.0025), T. philomelos (p=0.0271), F. coelebs (p<0.001), S. atricapilla (p=0.0190), and

−0.04

162.96 on 176 df

Residual deviance

*≤0.05, **≤0.01, ***<0.001

0.02

P1

4.30

*** 0.12

78.24 on 176 df

P1

3.35

***

***

−3.97 *** 3.19 **

z

0.001 −3.3

0.03

1.99 0.06

Estimate SE −5.15 *** −7.91 4.95 *** 0.19

z

0.001 −4.33 *** −0.005 102.47 on 176 df

0.62 −0.006

−0.52 0.59 0.12

1.44 0.04

Estimate SE

Mixd infections2

All mixed infections were between a Leucocytozoon parasite and either a Plasmodium or Haemoproteus one. We were not able to resolve mixed infections between Plasmodium and Haemoproteus, so mixed infections were probably underestimated (see also Santiago-Alarcon et al. 2011)

2

1

P1

Leucocytozoon

−1.19 0.23 −7.44 −1.52 0.12 0.23

z

0.001 0.49

131.88 on 176 df

<0.001

0.02

3.74

*** −0.01

Estimate SE 0.71 0.04

P1

Haemoproteus

−4.04 *** −0.85 2.87 ** −0.06

z

0.001 −3.03 **

0.03

1.98 0.05

91.03 on 176 df

*** 0.13

0.18

0.009 −4.73 *** −0.004

0.89

Body mass

Abundance * body mass

4.84

−5.19 *** −8.03 4.88 *** 0.17

Intercept Abundance

3.53 0.17

Estimate SE

−18.37 0.87

P1

Plasmodium

z

All data

Estimate SE

Variables

Table 2 Results from the general linear models (GLMs) for general prevalence (all data) and by each parasite genus in relation to bird abundance and body mass. We used a binomial distribution to fit models. df degrees of freedom

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marginally significant for E. rubecula (p=0.057). Parus major was the only species with a relatively high infection rate by Leucozytozoon, but it was not significantly higher than that of the other blood parasites (p=0.71). F. coelebs and S. atricapilla did not harbor any Leucozytozoon infections in our study. Juvenile T. merula and P. major had a significantly lower prevalence (28.5 and 45.45 % respectively) compared to adults (84.61 %, p=0.022 and 100 %, p<0.001, respectively). Finally, E. rubecula had an overall lower juvenile prevalence (4.54 %) compared to adults (23.07 %, p=0.0852).

Discussion In this study we present an analysis of the relative abundance of an urban avian community and its relationship to haemosporidian infection prevalence. Our results show that there is a significant positive relationship between infection status and body mass of an avian host. These results are in agreement with findings of previous studies for most understory bird species in Europe, in that larger bodied birds are expected to have a higher probability of infection (Scheuerlein and Ricklefs 2004). It is suggested that the mechanism behind this association is the larger amount of CO2 production of larger bodied animals, which is one of the main chemical cues used by blood-feeding insects to find their hosts (Klowden and Zweibel 2005); also, chemical cues on feathers have been found to be a vector attractant using experimental decoy birds, and these chemicals seem to produce more specificity than CO2 in regard to the vector species attracted (e.g. Weinandt et al. 2012). We also found a strong positive association between infection status and abundance. More abundant bird species are expected to have a higher infection rate simply because more individuals are expected to have a higher proportion of encounters with vectors (i.e. mass effect for frequency dependent transmission, Keeling and Rohani 2008). Heterogeneities of the environment also play an important role in infection risk; such as closeness to water bodies where vectors are more abundant (e.g. Wood et al. 2007; Knowles et al. 2014), and different forestry practices that can create puddles for vector breeding, which increases infection probabilities (Lüdtke et al. 2013). Although we found a significant interaction between body size and abundance, it seems that there is not a strong trade-off between these two variables in regard to infection probability. This suggests that vectors must use different mechanisms (e.g. such as size, chemical cues, host abundance), alone or in combination, in order to locate their host. For example, large hosts can be easily located by the larger amounts of chemical cues they release into the environment and also by their size (e.g. Weinandt et al. 2012), and small hosts can be located by a combination of their larger numbers (i.e. host availability) in the environment (Tempelis and Washino 1967; Richards et al. 2006) and chemical cues (Allan et al. 2006). An age-related effect on prevalence was detected in T. merula, P. major, and E. rubecula, where juveniles have a lower prevalence compared to adults. This pattern has been observed also in C. caeruleus from an England population (Wood et al. 2007; Knowles et al. 2011). However, Hatchwell et al. (2000) found no differences in blood parasite prevalence between juvenile and adult T. merula. Infections by haemosporidian parasites are expected to remain for a lifetime once the bird is initially infected (Valkiūnas 2005; Santiago-Alarcon et al. 2013b); hence, because exposure to vectors and parasites has been longer for adult birds compared to juveniles, it is expected that prevalence is higher for adults.

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When we conducted analyses separately for each parasite genus, we found that only Haemoproteus parasites were not associated to body mass and abundance of hosts. This might be related to the availability of vector families transmitting specific parasite genera and also to the availability/numbers of different parasite lineage genera to transmit at this urban forest, where the more common genetic lineages belong to the Haemoproteus genus (see Results). The vector community is dominated by Ceratopogonidae (Culicoides) vectors (SantiagoAlarcon et al. 2012a, 2013a), followed by Culicidae and Simuliidae species, which were very rare in our traps (Santiago-Alarcon pers. obs.). Because Culicoides vectors mostly transmit Haemoproteus parasites, which are the most commonly found haemosporidians at our study site, we suggest that both vector and parasite species are proportionally more common around the forest (Santiago-Alarcon et al. 2013a) compared to vectors transmitting other parasite genera (i.e. Culicidae-Plasmodium and Simuliidae-Leucocytozoon); therefore, location of potential hosts is not an issue limiting Haemoproteus parasite transmission based on the size and abundance of birds. In turn, rare vectors transmitting parasites, even generalist ones, belonging to less common genera need to undergo a more exhaustive search to locate their hosts, where using different cues that might be associated to host traits such as body mass (e.g. size, chemical cues) are essential for successful transmission. Alternatively, phylogenetically conserved host traits (e.g. immune systems), in particular at the family level (see SantiagoAlarcon et al. 2014), can determine parasite host range instead of host-vector encounters. In particular, Medeiros et al. (2013) showed that for some Plasmodium parasites transmitted by two generalist species of Culex mosquitoes in a local community of Chicago, bird hosts’ phylogenetic relatedness and no vector feeding preferences determine Plasmodium-bird associations. Evidence for this was provided by some common Plasmodium lineages (e.g. CHI02PL and CHI04PL) that comprised most of the infections in the sampled community, but that were rather restricted to one bird superfamily (Medeiros et al. 2013). However, this explanation might have less influence at the genus level because some of the most abundant bird species in the community (e.g. P. major, S. atricapilla, T. merula) belong to different families and all of them are infected by both Haemoproteus and Plasmodium parasites (Table 1). Thus, at the level of parasite genera, commonness of parasites from a genus in a community should mostly depend on competent generalist vector availability and less on host filtering traits (i.e. similar immune systems due to phylogenetic relatedness). We detected 43 different genetic lineages among the three haemosporidian genera in our summer sample. From these lineages, Plasmodium relictum genetic variants (e.g. P-SGS1) are the ones of major health concern for birds, as they have been shown to severely affect bird fitness (Møller and Nielsen 2007; Palinauskas et al. 2008, 2009, 2011) and are also known to infect a high number of bird species (e.g. Hellgren et al. 2009). Haemoproteus parasites dominated the parasite community; H. majoris (parasite commonly infecting blue and great tits), and H. prognei (a parasite infectious to house martins Delichon ubica) have been shown to negatively affect their hosts (Merino et al. 2000; Marzal et al. 2005; Martínez- de la Puente et al. 2010). In addition, H. minutus is a pathogenic widespread parasite, apparently specializing in species of the bird genus Turdus (Palinauskas et al. 2013), and we found the lineage HTURDUS2 infecting a large proportion of T. merula individuals in our study. It has been shown that generalist parasites have the highest prevalence in single host species (Hellgren et al. 2009); furthermore, haemosporidian genetic lineages that present high prevalence are known to infect a wider array of bird species (Szöllősi et al. 2011). Hence, we suggest that generalist parasites (e.g. P-SGS1, H-TURDUS2) using generalist vectors can increase their transmission success due to the wider array of potential hosts (e.g. Hellgren et al. 2009), where

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abundant and large (i.e. easily located) bird species represent some of the most valuable resources for both transmission and persistence of parasites. In Mooswald forest we have investigated Culicoides vectors (Santiago-Alarcon et al. 2012a, 2013a), which are implicated in the transmission of Haemoproteus parasites (Valkiūnas 2005; Santiago-Alarcon et al. 2012b). We have shown that most species of Culicoides, even those previously considered ornithophilic, are generalists taking blood meals from both mammals and birds (Santiago-Alarcon et al. 2012a, 2013a). This suggests that potentially any of the haemosporidian lineages, in particular common ones, detected in our study can be transmitted across a large array of bird species. Around cities people keep pet birds and zoos maintain species of conservation concern and some other exotic birds that can have latent infections when recently arrived (e.g. Murata et al. 2008; Pacheco et al. 2011; Paperna and Martelli 2008). Hence, it is advisable to undertake studies that address the possible host-parasite interplay between wildlife of natural urban reserves (e.g. parks) and urban animals (e.g. urban exploiters, pets, zoos) as a way of estimating potential risks for native birds and those under a conservation status. There is no consistent spatial pattern regarding infection prevalence in an urbanization gradient. While infection prevalence increases from rural to urban centers in some geographical locations (e.g. Belo et al. 2011; Evans et al. 2009), it decreases in cities compared to suburban or rural areas at other places (e.g. blood parasites, Bentz et al. 2006; Geue and Partecke 2008; Evans et al. 2009). Within the urban matrix there can be differences in levels of parasitism; for example, nests infected with Philornis porter had lower prevalence in parking lots compared to residential neighborhoods, and it was positively correlated with the amount of ground covered by buildings (LeGros et al. 2011). Hence, previous suggestions that urban areas provide a lower risk of haematozoan infections compared to rural/forested habitats and that this is a factor favoring invasion of urban ecosystems (Geue and Partecke 2008) is not warranted. This claim is also supported by the high avian haemosporidian richness identified at our studied urban forest. Urbanization effects on haemosporidian prevalence and intensity are rather species and context dependent, and they are particularly not well understood in tropical regions. At our study site prevalence was dominated by parasite infections of the genera Haemoproteus and Plasmodium compared to infections by parasites of the genus Leucocytozoon. This is a common pattern in bird species, where Haemoproteus and Plasmodium parasites generate the larger number of infections (Valkiūnas 2005). The blackcap is the best-studied species in relation to haemosporidian parasites in our sampling site, and in general across Europe (Pérez-Tris and Bensch 2005a, b; Pérez-Tris et al. 2007; Križanauskienė et al. 2010). In this study, we identified a 33.3 % infection for summer 2011, which is low compared to the 70.3 % mean prevalence observed during a 4-year period in the same population (Santiago-Alarcon et al. 2011). Infections were dominated by Haemoproteus parasites, followed by Leucocytozoon and Plasmodium parasites (Santiago-Alarcon et al. 2011). There is temporal variation in prevalence in this blackcap population (SantiagoAlarcon et al. 2011); hence, the low infection proportion of blackcaps for the summer of 2011 may simply reflect such inter-year or seasonal variability. This is rather surprising given that summer months are those with higher diversity and abundance of vectors (SantiagoAlarcon et al. 2013a), but it can reflect the stress that birds undergo during breeding months throughout the spring and not in summer when prevalence, as measured by using peripheral blood samples, decreases (e.g. Beaudoin 1971; Cosgrove et al. 2008; Santiago-Alarcon et al. 2011).

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Considering the limitations of our study due to a single sampling year during one single season, it would be advisable to include data from more years (longitudinal study) and also from the spring and autumn seasons, which are those where infection peaks are registered for some bird species (see above). Our work with insect vectors (i.e. Culicoides) at the Mooswald forest has showed us that the vector community is dynamic throughout the year (SantiagoAlarcon et al. 2013a), as such it would be expected that some parasite lineages are present in the bird community at the beginning (spring) but disappear later on, being replaced by other lineages that are transmitted by vector species that are present only at the end of the year. Thus, we can expect an increase in the number of haemosporidian lineages detected at this site when more years and seasons are included. Finally, if we consider the latitudinal diversity gradient (i.e. higher diversity at places closer to the equator), we can predict that comparable urban forests should hold a higher parasite species richness in tropical areas compared to the better known temperate urban forests. However, counterintuitive results (e.g. equal parasite host breadth between tropical and temperate areas) can occur given that parasites are not free living and evolution of their life cycles respond to different environmental factors (e.g. SvenssonCoelho et al. 2014).

Conclusion Taken together, our results and previous studies suggest that a more thorough understanding of parasite ecology (e.g. parasite host breadth) and transmission rates (e.g. vector competence for novel vector-parasite associations) in urban areas is necessary. During the last years it has been shown that parasites are essential components of a healthy ecosystem (Hudson et al. 2006; Lafferty et al. 2006, 2008), and that their influence on host dynamics can be profound and unpredictable under varied circumstances (Hatcher and Dunn 2011). Hence, ecosystem disturbances generated by the urbanization process can have unforeseen consequences for both animal and human health. In this sense, urban natural areas (e.g. protected parks) can play an important role in the conservation of both wildlife and their associated parasites (e.g. Whiteman and Parker 2005) on top of the numerous benefits that these green areas have for mental and physical health (e.g. Chiesura 2004; Tzoulas et al. 2007). Acknowledgments We thank Marie Melchior, Rebecca Bloch, Claudia Hermes, and Gregor Rolshausen for assistance during fieldwork. D.S.-A. was funded by the Alexander von Humboldt Foundation (post-doctoral grant) and by Consejo Nacional de Ciencia y Tecnología (CONACYT, project number CB-2011-01-168524). This work was also supported by the Deutsche Forschungsgemeinschaft (H.M.S., grant number 1008/6-1) and by the Wissenschaftliche Gesellschaft Freiburg (H.M.S. and G.S.). Conflict of interest The authors declare that they have no conflict of interest.

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