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Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Haemosporidians causing avian malaria are very common parasites among bird species. Their negative effects have been repeatedly reported in terms of deterioration in survival prospects or reproductive success. However, a positive association between blood parasites and avian fitness has also been reported. Here, we studied a relationship between presence of malaria parasites and reproductive performance of the host, a hole-breeding passerine – the blue tit Cyanistes caeruleus. Since the malaria parasites might affect their hosts differently depending on environmental conditions, we performed brood size manipulation experiment to differentiate parental reproductive effort and study the potential interaction between infection status and brood rearing conditions on reproductive performance. We found individuals infected with malaria parasites to breed later in the season in comparison with uninfected birds, but no differences were detected in clutch size. Interestingly, infected parents produced heavier and larger offspring with stronger reaction to phytohemagglutinin. More importantly, we found a significant interaction between infection status and brood size manipulation in offspring tarsus length and reaction to phytohemagglutinin: presence of parasites had stronger positive effect among birds caring for experimentally enlarged broods. Our results might be interpreted either in the light of the parasite-mediated selection or terminal investment hypothesis.

Parasites are ubiquitous and possibly most individuals face at least one infection episode during their lifetime. Infections with Haemosporidians, including avian malaria parasites, i.e. species from the genus Plasmodium and Haemoproteus (sensu Pérez-Tris et al. 2005), are commonly observed among birds. For example, among 74 surveyed species of European passerines 82% of bird species have been found to harbour Haemoproteus, and nearly 49% –Plasmodium (Scheuerlein and Ricklefs 2004). Malaria parasites are expected to negatively affect host health because they may damage the tissue of different organs including the liver, kidney and lungs (sporozoites) as well as to reduce the efficiency of the oxygen transport resulting from erythrocyte breakdown caused by gametocytes (Atkinson and van Riper 1991). Despite these harmful effects, the impact of avian malaria parasites on host's fitness is not uniform, but has rather been shown to vary across specific host–parasite systems (Palinauskas et al. 2008, Lachish et al. 2011). The most severe effects of malaria parasites have been demonstrated in the poultry, captive birds and naive populations, e.g. Hawaiian honeycreepers (Drepanidinae) (Atkinson et al. 2000). In wild populations these negative effects seem to be less severe and in many studies have not been detected at all (Valkiūnas 2005, Bensch et al. 2007), which is being attributed to the fact that the majority of individuals hold chronic infections characterized by low numbers of parasites in the blood (Valkiūnas 2005). The negative effects reported in wild populations include reduction in host body condition (Merino et al. 2000, Valkiūnas et al. 2006), lower reproductive success (Merilä and Andersson 1999, Asghar et al. 2011) and survival (Valkiūnas 2005, Martínez-de la Puente et al. 2010). Interestingly, the number of studies showed that host performance may also be positively associated with infection status. Such a positive association has been reported in case of clutch size (Sanz et al. 2001, Fargallo and Merino 2004), fledging success (Norte et al. 2009) and offspring survival (Kilpatrick et al. 2006).

Here, we aimed at studying whether the status of infection with malaria parasites is associated with reproductive performance in a small passerine, the blue tit. Previous studies showed that in this species malaria parasites may negatively affect host reproductive performance in terms of hatching and fledging success (Merino et al. 2000, Knowles et al. 2010a) or nestling condition (Merilä and Andersson 1999, Knowles et al. 2010a). Here, however we explored whether the potential impact of parasite infection might be diversely expressed under different environmental conditions simulated by experimental brood size manipulation. Brood size enlargement has been shown to result in increased parasitemia (Knowles et al. 2009, 2010b), which is usually attributed to down regulation of defence mechanism in face of increased reproductive demands (Merilä and Andersson 1999, Knowles et al. 2009). However, the resolution of this trade-off may not be such simple and should depend on infection status of the parents, but this aspect has rarely been studied. Thus, here we employed brood size manipulation experiment to increase reproductive effort of the parents and we predicted that the negative association between parasitic infections and reproductive performance should be more strongly pronounced among parents forced to rear enlarged broods than in parents caring for unmanipulated broods.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The study was conducted from 2008 to 2011 on the island of Gotland, Sweden (57°03′N, 18°17′E) in the population of blue tits breeding in nest boxes. The first clutch contains on average 11 eggs. Chicks hatch after two weeks of incubation and fledge 18–22 d later. Study area consists of a dozen wood plots separated by arable fields. For detailed description of the study site see Przybylo et al. (2000).

From the end of April, nest-boxes were regularly inspected to record the date of egg laying, the number of eggs and the date of hatching (day = 0). To create two types of broods differing in the level of parental effort (with unmanipulated, henceforth control, and enlarged brood size), two broods of similar size (± 1 nestling) and the same hatching date were paired two days post-hatching and one randomly chosen brood from a pair received three extra nestlings. These extra nestlings were of similar weight as those in a recipient brood and originated from a donor nest not involved in the experiment. In total, there were 43 control and 53 enlarged broods. Control and enlarged broods did not differ prior to manipulation in the clutch size (control versus enlarged (mean ± SD): 10.77 ± 1.17 vs 10.91 ± 1.42, t = 0.51, DF = 94, p = 0.61) and the number of hatchlings (9.05 ± 1.56 vs 9.09 ± 1.64, t = 0.15, DF = 94, p = 0.89). There was a difference in the number of nestlings on day 14 post-hatching (8.51 ± 2.07 vs 12.02 ± 1.65, t = 9.24, DF = 94, p < 0.0001). Additionally, for the purpose of another experiment, half of the nestlings was swapped between paired broods (control-enlarged) on the same day when the brood size was manipulated. Nestlings were uniquely marked by nail clipping two days after hatching, and ringed on day 11 after hatching. On the same day (11) their immune function was assessed with phytohemagglutinin (PHA) test. Phytohemagglutinin (PHA) is a non-pathogenic antigen, which is widely used in ecological research to measure cell-mediated immune response (Pickett et al. 2013). Subcutaneous injection of PHA causes local swelling by accumulation of cells representing both adaptive and innate immunity (Martin et al. 2006, Vinkler et al. 2012). Nestlings were injected with saline solution containing 0.2 mg of PHA (Sigma Aldrich, Poznan, Poland) into the right wing web. The thickness of the wing web was measured before and 24 ± 2 h after the injection using pressure-sensitive dial thickness gauge (Mitutoyo, Tokyo, Japan). All measurements in each year were taken by the same person in triplicate for the given nestling and were highly repeatable (Drobniak et al. 2010). The level of immune response was calculated as a difference between mean value of wing web thickness before and after the injection (Smits et al. 1999). On day 14 after hatching nestlings were weighted with an electronic balance (to the nearest 0.1 g) and had their tarsus length measured with an electronic caliper (to the nearest 0.01 mm).

Adult birds were captured, either in the nest box with traps or by mist-netting, while feeding 14 d old nestlings. If catching attempt was unsuccessful on that day, it was repeated the following days. Birds were aged as yearlings or as at least 2 yr old based on the colour of the wing coverts or according to the ringing records and sexed by presence/absence of the brood-patch (Svensson 1994). Adults were bled from the wing vein using capillary and samples were stored in room temperature in 96% ethanol. DNA was extracted using Chelex (Bio-Rad, Munich, Germany) following the manufacturer's protocol (Walsh et al. 1991). Samples were screened for the presence of blood parasites (genus Haemoproteus and Plasmodium) by amplifying a 478 bp fragment of the mitochondrial cyt b gene, using nested polymerase chain reaction (Waldenström et al. 2004). PCR reactions were performed in 25 μl volumes, in two separate runs as described by Cosgrove et al. (2008). Four μl of PCR products from the second round were run on 2% agarose gels stained with GelRed (Biotium, Hayward, CA, USA) and visualized under ultraviolet light. Each plate contained the positive (DNA from individuals with confirmed infection based on blood smear screening) and negative (ddH2O) control, to control for possible contamination or failures during PCRs. The quality of DNA isolate of all samples which yielded no PCR product, was checked with sex-specific primers (Griffiths et al. 1998). PCR products of positive samples were purified with FastAP (Fermentas) and then sequenced directly with an automated ABI 3130 DNA analyzer (Applied Biosystems) using BigDye terminator ver. 3.1 (Applied Biosystems). The obtained sequences were aligned and compared with the MalAvi database (Bensch et al. 2009) using BioEdit software (Hall 1999). Mixed infection were rare (less than 4% of infections) and assessed according to the occurrence of double peaks in the chromatogram. Cases of mixed infections were excluded from the analyses. The studied population is sedentary, so blood parasites seem to be locally transmitted. We assume that positive infection status corresponds to chronic infection, because it is rather uncommon to find individuals with acute stage of infection in nature (Lachish et al. 2011). However, we do not have data to confirm this.

Variation in clutch size and time of breeding in relation to infection status of the parents was analyzed using general linear mixed model, fitted in SAS 9.3. The model included infection status of adults (treated as a three-level factor: both parents uninfected (BU), only one parent infected (OI), both parents infected(BI)) and the year of the study defined as a random factor. These analyses are based on all broods from the population for which infection status of both parents is known (in total 220 clutches). Further analyses were conducted on smaller sample size – only clutches involved in brood size experiment were considered (n = 96).

To analyse the effect of experimental treatment, age and sex on the prevalence of blood parasites we performed generalised linear mixed models with a logit link function assuming a binomial error, fitted in SAS 9.3. The full model included above variables as fixed factors and their interactions and then non-significant interactions were sequentially removed. The effect of the infection status of rearing parents on nestling's body mass, tarsus length and the strength of immune response was estimated with the general linear mixed model, fitted in SAS 9.3. The response to PHA was log-transformed to meet the assumption of normal distribution. The model included infection status of rearing parents (treated as a three-level factor: both parents uninfected (BU), only one parent infected (OI), both parents infected (BI)), the experimental treatment (enlarged or control brood) and their interaction as fixed factors. The model included random factors: year of the study, nest of origin, nest of rearing and block (paired nests) as a higher level categorical variables. Block accounts for variation in timing of breeding and clutch size. Full model was always tested, but then reduced by removing non-significant interactions. Because older individuals and those breeding later in the season were more likely to carry parasitic infection (see the Results section), in the preliminary analyses we additionally considered parental age defined as a three-level factor (BY – both parents young, OY – one parent young and another old, and BO – both parents old) and laying date defined as a continues covariate. However these factors and the interaction of these factors with other variables in the model appeared non-significant. So we decided to discard these effects from the final model. In the analyses we used only broods of known infection status of both parents. In total 843 nestlings from 96 nests were included in the analyses. The differences between groups were analysed using post-hoc Tukey test. The sample size of each group with respect to infection status of parents was as follows: both uninfected – 21 pairs, only one parent infected – 31 pairs and both infected – 44 pairs. Much higher frequency of the last group results from a generally high prevalence of malaria parasites in the study population (63% of individuals in the population carry infection with malaria parasites, own unpublished data). During 4 yr of study there were three females and four males breeding in two seasons and one male in three seasons, but in each case these individuals were paired with different partner, so these birds were not excluded from analyses.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The prevalence of infection with malaria parasites among birds involved in experimental treatment (individuals caring for control and enlarged broods) was 62% (119/192). Most birds were infected with parasites from the genus Plasmodium (71 vs 29% infected with parasites from the genus Haemoproteus; Supplementary material Appendix 1, Table A1). Parents caring for control and enlarged broods did not differ in the prevalence of malaria parasites (59 vs 65%, χ12= 0.99, p = 0.32). The prevalence of blood parasites did not differ between sexes (60% infected females vs 65% males, χ12= 0.33, p = 0.56), but differed between age classes: in yearling birds 55% of individuals were infected, while in older birds 70% were infected (χ12= 4.84, p = 0.03).

We found a significant relationship between infection status and time of breeding. Laying date was significantly delayed when both partners within a pair were infected in comparison with pairs, in which either both parents were uninfected or only one was infected (mean ± SD, 1 April = day 1, laying date: BI: 29.84 ± 0.52, BU: 28.18 ± 0.79, OI: 28.19 ± 0.54, F2,169= 3.69, p = 0.03). There were no differences in clutch size between groups representing various infection status (F2,213= 0.35, p = 0.70), although clutch size was significantly affected by laying date (F1,188= 5.55, p = 0.02). However, the interaction between laying date and infection status was not significant (F1,174= 0.22, p = 0.80).

We found a significant interaction between parental infection status and experimental treatment in the analyses of variation in nestlings’ tarsus length and reaction to PHA, suggesting that parental infection status differently affected tarsus length and swelling reaction of the nestlings originating from enlarged and control broods (Table 1). Post-hoc analyses (Tukey test) revealed that among enlarged broods parental infection status was positively associated with nestling tarsus (BU vs OI: p = 0.001; BU vs BI: p = 0.001) and response to PHA (BU vs OI: p = 0.003; BU vs BI: p = 0.003), while such effect was not observed among control broods (Fig. 1, 2). In case of body mass the interaction was not significant, but both infection status and experimental treatment appeared significant (Table 1). The post-hoc test showed that nestlings from broods attended by both infected parents (BI) had larger body mass than nestlings reared by both uninfected parents (BU) (Tukey HSD, p < 0.001, Fig. 3) and ones from enlarged broods were lighter than those from control broods (Tukey HSD, p < 0.001).

Table 1. General linear mixed model analyzing variation in nestling PHA response, nestling body mass and tarsus length. Significant terms (p < 0.05) are shown in bold. Non-significant interactions were discarded from the model. The model included also random factors: year of the study, nest of origin, nest of rearing and block (paired nests) – effects not shown.
Response variablePredictorsDFDen DFF-valuePr > F
Nestlings PHA responseExperimental treatment181.48.86 0.004
Infection status285.12.170.121
Infection status × Experimental treatment289.44.02 0.021
Nestlings tarsus on the 14 dExperimental treatment163.89.87 0.002
Infection status281.35.34 0.007
Infection status × Experimental treatment292.74.98 0.009
Nestlings body mass on the 14 dExperimental treatment148.617.75 0.0001
Infection status265.27.61 0.001
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Figure 1. Effect of parental infection status on nestling tarsus length on day 14 in unmanipulated broods (filled circles, solid line) and enlarged broods (open squares, dashed line). Points present least sq means and standard errors.

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image

Figure 2. Effect of parental infection status on nestling immune response in unmanipulated broods (filled circles, solid line) and enlarged broods (open squares, dashed line). Points present least sq means and standard errors.

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image

Figure 3. Effect of parental infection status on nestling body mass on day 14 in unmanipulated broods (filled circles, solid line) and enlarged broods (open squares, dashed line). Points present least sq means and standard errors.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We showed that blue tits infected with malaria parasites breed later in the season. This might be considered as a negative effect of parasites since reproductive performance generally declines with the advancement of the season (Perrins 1991, Nilsson 2000), possibly due to seasonal deterioration in food availability (Daan et al. 1988). On the other hand, it is also possible that parasitic infection is a result, not a cause, of later breeding. If vector abundance increases with the season progress individuals breeding later in season may show higher probability of acquiring new infections (Cosgrove et al. 2008). Unfortunately, we are not able to test these alternatives. Interestingly, contrary to some common intuition assuming negative impact of parasites on individual performance, we showed that infected parents raised nestlings showing higher condition than nestlings of uninfected parents. Specifically, nestlings from broods reared by infected parents were larger and showed stronger reaction to PHA than those reared by uninfected parents.

The majority of previous studies based on correlational data demonstrated either the lack of effect or a negative effect of malaria parasites on reproductive performance (Bensch et al. 2007, Ortego et al. 2008, Asghar et al. 2011). Additionally, recent studies employing the experimental reduction of infection with malaria parasites in wild populations have shown that these parasites may exert much stronger negative effects on host fitness than previously assumed. Birds treated with anti-malarial drugs not only showed elevated reproductive performance, but also higher survival rate (Merino et al. 2000, Knowles et al. 2010a, b, Martinez-de la Puente et al. 2010). Thus, the negative effect of parasitic infection is clearly expected. However, the positive association between infection status and reproductive performance has been also observed across diverse taxa (Minchella and LoVerde 1981, Schwanz 2008), including birds (Sanz et al. 2001, Marzal et al. 2008). Our study also shows a positive relationship between infection status and reproductive performance. We found that the infection with malaria parasites was much strongly related to reproductive investment as reflected by longer tarsus and stronger swelling reaction among parents caring for experimentally enlarged than for control broods. Clearly, brood size enlargement appeared to have weaker effect on nestlings raised by parasitized parents in comparison with nestlings of uninfected parents. This result is quite unexpected, and difficult to explain. The most likely explanation of such effect is that infected parents must have enhanced their reproductive effort more than uninfected parents in face of increased reproductive demands. Relatively better performance of infected parents may potentially be explained by differential survival of infected individuals. It is possible that infected individuals observed in the population have survived an acute stage of infection and are able to control the infection intensity at the low level as suggested by Westerdahl et al. (2012). The individuals surviving infection may in fact be of superior quality and therefore they may better cope with increased feeding demands invoked by our brood size manipulation in comparison with uninfected individuals. This explanation is in line with the so called parasite-mediated selection (Goater and Holmes 1997).

Another potential mechanism explaining the observed positive association between parasitic infection and reproductive performance may be the terminal investment hypothesis (Williams 1966), which assumes that an individual should invest more into current reproduction in face of reduced prospects of future reproduction. Unfortunately, our data do not allow to disentangle between these alternative explanations. It would require large data set allowing rigorous survival analyses. Demonstrating the parasite-mediated selection hypothesis would require showing that only individuals in prime condition survive infection leading to significant differences in individual quality between infected and uninfected individuals. Such parasite-mediated selection should also affect variance observed between infected and uninfected individuals. The terminal investment hypothesis requires showing that parasitic infection strongly reduces life expectancy, making future prospects of breeding unlikely. To our knowledge, support for the parasite-mediated selection hypothesis based on rigorous survival analyses does not exist and the terminal investment hypothesis has not received convincing support rooted in survival analyses coupled with reproductive performance. Recent studies on blue tits provide some evidence that malaria parasites may negatively affect the probability of survival in this species (Stjernman et al. 2008, Martinez-de la Puente et al. 2010, Lachish et al. 2011). Specifically, Martinez-de la Puente et al. (2010) showed that the reduction in the intensity of infection with parasites from the genus Haemoproteus in blue tit females due to drug treatment was associated with their higher survival to following breeding seasons, while Stjernman et al. (2008) showed that survival in this species is affected by the intensity of infection with Haemoproteus in a non-linear manner with the highest survival in birds harboring the intermediate parasitemia. However, Lachish et al. (2011) demonstrated on a large data set that the overall impact of infection with malaria parasites on blue tit survival rates was rather small and it depended on the parasite lineage.

In conclusion, we showed the positive association between infection with malaria parasites and reproductive performance in blue tits. Our study suggests that challenging breeding conditions, simulated here by experimentally increased reproductive effort, may constitute an important factor affecting potential relationship between blood parasites and host reproductive performance. Although both the terminal investment hypothesis and the parasite-mediated selection hypothesis are very attractive and possible explanations of a positive relationship between infection status and reproductive performance, our data are not suitable to provide a sufficient support for neither of them.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We would like to thank Dariusz Wiejaczka for assistance with the fieldwork. Financial support was provided by the National Science Centre, grant no. N N304 336039 to AD. The long term nest box study was supported by the Swedish Research Council (grant to LG). The study conforms to the legal requirements of Sweden.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Asghar M. , Hasselquist D. and Bensch S . 2011 . Are chronic avian haemosporidian infections costly in wild birds?J. Avian Biol. 42 : 530537 .
  • Atkinson C. T. and van Riper III , C . 1991 . Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon, and Haemoproteus . – In: Loye J. E. and Zuk M. (eds), Bird–parasite interactions . Oxford Univ. Press , pp. 1948 .
  • Atkinson C. T. , Dusek R. J. , Woods K. L. and Iko W. M . 2000 . Pathogenicity of avian malaria in experimentally-infected Hawaii Amakihi . – J. Wildl. Dis. 36 : 197204 .
  • Bensch S. , Waldenström J. , Jonzén N. , Westerdahl H. , Hansson B. , Sejberg D. and Hasselquist D . 2007 . Temporal dynamics and diversity of avian malaria parasites in a single host species . – J. Anim. Ecol. 76 : 112122 .
  • Bensch S. , Hellgren O. and Perez-Tris J . 2009 . MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages . – Mol. Ecol. Resour. 9 : 13531358 .
  • Cosgrove C. L. , Wood M. J. , Day K. P. and Sheldon B. C . 2008 . Seasonal variation in Plasmodium prevalence in a population of blue tits Cyanistes caeruleus . – J. Anim. Ecol. 77 : 540548 .
  • Daan S. , Dijkstra C. , Drent R. and Meijer T . 1988 . Food supply and the annual timing of avian reproduction . – Proc. Int. Ornithol. Congr. 19 : 392407 .
  • Drobniak S. M. , Wiejaczka D. , Arct A. , Dubiec A. , Gustafsson L. and Cichoń M. 2010 . Sex-specific heritability of cell-mediated immune response in the blue tit nestlings (Cyanistes caeruleus) . – J. Evol. Biol. 23 : 12861292 .
  • Fargallo J. A. and Merino S . 2004 . Clutch size and haemoparasite species richness in adult and nestling blue tits . – Ecoscience 11 : 168174 .
  • Goater C. P. and Holmes J. C . 1997 . Parasite-mediated natural selection . – In: Clayton D. H. and Moore J. (eds), Host–parasite evolution: general principles and avian models . Oxford Univ. Press , pp. 929 .
  • Griffiths R. , Double M. , Orr K. and Dawson R . 1998 . A DNA test to sex most birds . – Mol. Ecol. 7 : 10711075 .
  • Hall T . 1999 . BioEdit. Biological sequence alignment editor for Windows . – North Carolina State Univ., NC, USA , < www.mbio.ncsu.edu/BioEdit/bioedit.html >.
  • Kilpatrick A. M. , LaPointe D. A. , Atkinson C. T. , Woodworth B. L. , Lease J. K. , Reiter M. E. and Gross K . 2006 . Effects of chronic avian malaria (Plasmodium relictum) infection on reproductive success of Hawaii Amakihi (Hemignathus virens) . – Auk 123 : 764774 .
  • Knowles S. C. L. , Nakagawa S. and Sheldon B. C . 2009 . Elevated reproductive effort increases blood parasitaemia and decreases immune function in birds: a meta-regression approach . – Funct. Ecol. 23 : 405415 .
  • Knowles S. C. L. , Palinauskas V. and Sheldon B. C . 2010a . Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population . – J. Evol. Biol. 23 : 557569 .
  • Knowles S. C. L. , Wood M. J. and Sheldon B. C . 2010b . Context-dependent effects of parental effort on malaria infection in a wild bird population, and their role in reproductive trade-offs . – Oecologia 164 : 8797 .
  • Lachish S. , Knowles S. C. L. , Alves R. , Wood M. J. and Sheldon B. C . 2011 . Fitness effects of endemic malaria infections in a wild bird population: the importance of ecological structure . – J. Anim. Ecol. 80 : 11961206 .
  • Martin L. , Han P. , Lewittes J. , Kuhlman J. R. , Klasing K. C. and Wikelski M . 2006 . Phytohemagglutinin-induced skin swelling in birds: histological support for a classic immunoecological technique . – Funct. Ecol. 20 : 290299 .
  • Martínez-de la Puente J. , Merino S. , Tomás G. , Moreno J. , Morales J. , Lobato E. , García-Fraile S. and Belda E. J . 2010 . The blood parasite Haemoproteus reduces survival in a wild bird: a medication experiment . – Biol. Lett. 6 : 663665 .
  • Marzal A. , Bensch S. , Reviriego M. , Balbontin J. and de Lope F . 2008 . Effects of malaria double infection in birds: one plus one is not two . – J. Evol. Biol. 21 : 979987 .
  • Merilä J. and Andersson M . 1999 . Reproductive effort and success are related to haematozoan infections in blue tits . – Ecoscience 6 : 421428 .
  • Merino S. , Moreno J. , Sanz J. J. and Arriero E . 2000 . Are avian blood parasites pathogenic in the wild? A medication experiment in blue tits (Parus caeruleus) . – Proc. R. Soc. B 267 : 25072510 .
  • Minchella D. J. and LoVerde P. T . 1981 . A cost of increased early reproductive effort in the snail Biomphalaria glabrata . – Am. Nat. 118 : 876881 .
  • Nilsson J.-Å. 2000 . Time-dependent reproductive decisions in the blue tit . – Oikos 88 : 351361 .
  • Norte A. C. , Araújo P. M. , Sampaio H. L. , Sousa J. P. and Ramos J. A . 2009 . Haematozoa infections in a great tit Parus major population in central Portugal: relationships with breeding effort and health . – Ibis 151 : 677688 .
  • Ortego J. , Cordero P. J. , Aparicio J. M. and Calabuig G . 2008 . Consequences of chronic infections with three different avian malaria lineages on reproductive performance of lesser kestrels (Falco naumanni) . – J. Ornithol. 149 : 337343 .
  • Palinauskas V. , Valkiūnas G. , Bolshakov C. V. and Bensch S . 2008 . Plasmodium relictum (lineage P-SGS1): effects on experimentally infected passerine birds . – Exp. Parasitol. 120 : 372380 .
  • Pérez-Tris J. , Hasselquist D. , Hellgren O. , Krizanauskiene A. , Waldenström J. and Bensch S . 2005 . What are malaria parasites?Trends Parasitol. 21 : 209211 .
  • Perrins C. M . 1991 . Tits and their caterpillar food supply . – Ibis 133 (Suppl. 1) : 4954 .
  • Pickett S. R. A. , Weber S. B. , McGraw K. J. , Norris K. J. and Evans M. R . 2013 . Environmental and parental influences on offspring health and growth in great tits (Parus major) . – PLoS One 8 : e69695 .
  • Przybylo R. , Sheldon B. C. and Merilä J. 2000 . Climatic effects on breeding and morphology: evidence for phenotypic plasticity . – J. Anim. Ecol. 69 : 395403 .
  • Sanz J. J. , Arriero E. , Moreno J. and Merino S . 2001 . Interactions between hemoparasite status and female age in the primary reproductive output of pied flycatchers . – Oecologia 126 : 339344 .
  • Scheuerlein A. and Ricklefs R. E . 2004 . Prevalence of blood parasites in European passeriform birds . – Proc. R. Soc. B 271 : 13631370 .
  • Schwanz L. E . 2008 . Chronic parasitic infection alters reproductive output in deer mice . – Behav. Ecol. Sociobiol. 62 : 13511358 .
  • Smits J. E. , Bortolotti G. R. and Tella J. L . 1999 . Simplifying the phytohaemagglutinin skin-testing technique in studies of avian immunocompetence . – Funct. Ecol. 13 : 567572 .
  • Stjernman M. , Råberg L. and Nilsson J.-Å. 2008 . Maximum host survival at intermediate parasite infection intensities . – PLoS One 3 : e2463 .
  • Svensson L . 1994 . Identification guide to European passerines . – Stockholm .
  • Valkiūnas G . 2005 . Avian malaria parasites and other Haemosporidia . – CRC Press .
  • Valkiūnas G. , Žičkus T. , Shapoval A. P. and Iezhova T. A . 2006 . Effect of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) on body mass of the blackcap Sylvia atricapilla . – J. Parasitol. 92 : 11231125 .
  • Vinkler M. , Schnitzer J. , Munclinger P. and Albrecht T . 2012 . Phytohaemagglutinin skin-swelling test in scarlet rosefinch males: low-quality birds respond more strongly . – Anim. Behav. 83 : 1723 .
  • Waldenström J. , Bensch S. , Hasselquist D. and Östman Ö. 2004 . A new nested PCR method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood . – J. Parasitol. 90 : 191194 .
  • Walsh P. S. , Metzger D. A. and Higuchi R . 1991 . Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material . – BioTechniques 10 : 506513 .
  • Westerdahl H. , Asghar M. , Hasselquist D. and Bensch S . 2012 . Quantitative disease resistance: to better understand parasite-mediated selection on major histocompatibility complex . – Proc. R. Soc. B 279 : 577584 .
  • Williams G. C . 1966 . Natural selection, the costs of reproduction, and a refinement of Lack's principle . – Am. Nat. 100 : 687690 .

Supplementary material (Appendix JAV-00284 at < www.oikosoffice.lu.se/appendix >). Appendix 1.