Journal of Ornithology

, Volume 149, Issue 3, pp 337–343

Consequences of chronic infections with three different avian malaria lineages on reproductive performance of Lesser Kestrels (Falco naumanni)


    • Grupo de Investigación de la Biodiversidad Genética y CulturalInstituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM)
  • Pedro J. Cordero
    • Grupo de Investigación de la Biodiversidad Genética y CulturalInstituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM)
  • José Miguel Aparicio
    • Grupo de Investigación de la Biodiversidad Genética y CulturalInstituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM)
  • Gustau Calabuig
    • Grupo de Investigación de la Biodiversidad Genética y CulturalInstituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM)
Original Article

DOI: 10.1007/s10336-008-0287-9

Cite this article as:
Ortego, J., Cordero, P.J., Aparicio, J.M. et al. J Ornithol (2008) 149: 337. doi:10.1007/s10336-008-0287-9


We studied the consequences of chronic infections by three different lineages of avian malaria, two Plasmodium (RTSR1, LK6) and one Haemoproteus (LK2), on reproductive performance of Lesser Kestrels (Falco naumanni). Malaria infections in male and female parents had no effect on clutch size, hatching success or nesting success. However, when only successful nests were considered, we found that males parasitized by LK6 raised a lower number of fledglings, suggesting that the level of parental effort by males may be limited by this particular lineage of Plasmodium. This effect was not evident in females, probably due to the higher investment of males during the chick rearing period in this species. Overall, we have found that chronic stages of specific malaria lineages have certain negative consequences on host reproductive performance, highlighting the importance of considering genetic differences among malaria parasites to study their consequences on natural bird populations.


Avian malariaDiseaseFalco naumanniHaemoproteusPlasmodium


Infectious diseases have a major influence on host population dynamics and evolution, promoting host genetic variation and influencing co-evolutionary processes (Hamilton and Zuk 1982; Poulin et al. 2000). The study of the interaction between avian malaria parasites and their hosts has greatly contributed to the understanding of several aspects of host/parasite ecology and evolution (Hamilton and Zuk 1982; Poulin et al. 2000; Bensch et al. 2000). Further, avian malaria parasites are responsible for widespread declines in Hawaiian native forest birds (van Riper et al. 1986), and several negative effects, such as increased mortality and reduced reproductive outputs, have been described in a number of bird species (Atkinson et al. 2000; Dawson and Bortolotti 2000; Merino et al. 2000; Sol et al. 2003). For this reason, researchers have devoted much effort to studying these parasites and their consequences on natural bird populations.

In recent years, DNA sequencing has revealed several cases of cryptic speciation of avian malaria, indicating that the number of species in this parasite group is much higher than previously thought (Bensch et al. 2000, 2004; Fallon et al. 2005; Ricklefs et al. 2005). Thus, several species of avian malaria, originally defined based on host taxonomy or morphological criteria, have been found to be composed of a number of independent evolutionary units (Bensch et al. 2000, 2004; Beadell et al. 2006). After the application of molecular techniques, the study of avian malaria has switched from considering variation at the level of genera to analyzing specific lineages defined on the basis of molecular information (Bensch et al. 2004, 2007). This has important applied and theoretical implications because different lineages may have evolved different adaptations and affect host differentially (Westerdahl et al. 2005). Previous studies analyzing data obtained on the basis of visual examination of blood smears are likely to have disregarded and/or confounded different and potentially pathological effects of cryptic lineages. Furthermore, recent studies have shown that several malaria lineages are undetected by traditional visual blood smear analyses, indicating that the prevalence of malaria has been severely underestimated (Waldenström et al. 2004; Ortego et al. 2007a). However, although molecular techniques have now been widely applied to the study of avian malaria parasites, to the best of our knowledge only a single study has analyzed the consequences on host fitness of different molecularly typed lineages in a wild bird population (Bensch et al. 2007).

The Lesser Kestrel (Falco naumanni) is a colonial and migratory falcon. This species was once one of the most abundant birds of prey in southern Europe, but suffered a sharp population decline in its Western Paleartic breeding range in the second half of the twentieth century that led to complete extinction in several countries and to strong declines in others (Biber 1990). Thus, it is of great interest to analyze the potential negative consequences of avian malaria parasites on this species. A previous study has reported that infections by Haemoproteus parasites had no effect on clutch size in this species, but the potential consequences of malaria parasites on other parameters related to the species reproductive performance have not so far been analysed (Tella et al. 1996). Also, that study was based on visual examinations of blood smears, and parasites of the genera Plasmodium, supposedly the most pathogenic ones (Bensch et al. 2000; Atkinson et al. 2000), probably went undetected because they occurred at intensities of infection that are hard to detect in blood smears (Ortego et al. 2007a).

Molecular techniques have now revealed that Lesser Kestrels are parasitized by at least six different avian malaria lineages, with two Plasmodium (RTSR1, LK6) and one Haemoproteus (LK2) lineages being the most common in our study population from Central Spain (Ortego et al. 2007a). The mean prevalence of these three lineages in the study population is 3.4% for RTSR1, 4.6% for LK6 and 4.1% for LK2; no mixed infections have been detected (Ortego et al. 2007a). According to the obtained sequence divergences (RTSR1–LK6: 6.9%; RTSR1–LK2: 12.8%; LK2–LK6: 14.9%), these lineages seem to correspond to three well-differentiated species (Ortego et al. 2007a), and so they may have evolved different adaptations and affect hosts differentially (Bensch et al. 2004). At least one of these lineages (RTSR1) has active transmission in Africa as it has been also detected in African resident birds coexisting with the Lesser Kestrel in its wintering area (Waldenström et al. 2004; Ortego et al. 2007a). On the other hand, there is no evidence of transmission of these parasites in the European breeding quarters as they have not been found infecting fledgling Lesser Kestrels sampled in northeastern (n = 85; Tella et al. 1996) and central Spain (n = 279; J. Ortego et al., unpublished). Infection intensities of these lineages in breeding Lesser Kestrels are generally below the limit of detection by methods based on microscopy examinations of blood smears (Ortego et al. 2007a), indicating low-intensity peripheral parasitemia characteristic of chronic infections (Jarvi et al. 2003). Thus, avian malaria lineages parasitizing adult Lesser Kestrels on breeding grounds probably circulate in the blood stream as chronic infections. In the present study, we used blood parasite data reported in a previous study (Ortego et al. 2007a) to investigate the consequences of chronic infections by these malaria lineages on the reproductive performance of Lesser Kestrels.


Study population and field procedures

The study was conducted in La Mancha, central Spain (600–800 m asl), in an area covering approximately 1,000 km2. We studied 30 Lesser Kestrel colonies clustered in two subpopulations separated by 30 km: “Villacañas” subpopulation (39°30′N, 3°20′W; 24 colonies) and “Consuegra” subpopulation (39°35′N, 3°40′W; 6 colonies). The climate is meso-Mediterranean with mean temperatures ranging from 24–26°C in July to 4–6°C in January, and with 300–400 mm of rainfall mainly concentrated in spring and autumn.

In our study area, the Lesser Kestrel mainly breeds in abandoned farmhouses, both under tiled roofs and inside holes in walls. Kestrels normally arrive in this area from their winter quarters in Africa in mid-February or the beginning of March, depending on the year. Egg laying lasts from the end of April to the first week of June (Aparicio and Bonal 2002). Kestrels are mainly monogamous, and lay only one clutch per season with the exception of rare replacement clutches (ca. 0.5%). During the 2001–2006 breeding seasons, we located nest-sites before the onset of egg laying. Each potential nest was monitored every 6 days from the middle of April to find the first eggs, and then every 2 days until the clutch was finished. Thus, since females usually lay four or five eggs with mean intervals of 2 days, we were able to find the maximum number of unfinished clutches with minimum disturbance of the colonies. Laying date was defined as the date the first egg was laid (Aparicio and Bonal 2002). Near the expected hatching dates, we inspected the nests regularly to ascertain the exact hatching pattern of the eggs. We defined as unhatched eggs those that survived the entire incubation period but failed to hatch. Young were marked at hatching with a waterproof felt-tip pen, and they were banded 5–7 days later (for more details on field methods, see Aparicio 1997).

Adults were trapped with a noose carpet or by hand during incubation, measured and individually marked with metallic and colored plastic rings. Blood samples (100 μl) were obtained by venipuncture of the brachial vein and preserved in ∼1,200 μl ethanol 96% at −20°C. We used pectoral thickness as an estimator of adult body condition (Aparicio 1997; Aparicio and Cordero 2001). This trait has been used in previous studies as a measure of body condition in several bird species (Bolton et al. 1991; Newton 1993), and has been considered a more reliable measure of condition than residuals of body mass on tarsus length (Gosler and Harper 2000). Moreover, it is easy to measure accurately on live birds by using a portable ultrasonic meter, in this case a Krautkrämer USM22F (accuracy 0.1 mm), specially designed to measure animal tissues. We knew the exact age of around a third of individuals that were ringed as fledglings. For all other birds, we considered that individuals captured for the first time were in their first year if they presented yearling plumage or in their second year if they presented adult plumage (e.g., Aparicio and Cordero 2001; Foerster et al. 2003). This approach is not likely to bias age estimates because modal life-span in Lesser Kestrels is only 4 years and almost all nestlings and ∼70% of breeding birds are captured and banded every year in the study area (Ortego et al. 2007b).

Screening for avian malarial infections

We used QIAamp DNA Blood Mini Kits (QIAGEN) to extract and purify DNA from the blood samples. Each bird was screened for malaria infection using a highly efficient nested PCR protocol that amplifies a 524-bp fragment (including primers) of the mitochondrial cytochrome b gene of both Plasmodium and Haemoproteous parasites (Waldenström et al. 2004). This method consists of two rounds, an initial 20 cycles of PCR using the primers HAEMNF and HAEMNR2 and a final 35 cycles of PCR using the internally nested primers HAEMF and HAEMR2 (Waldenström et al. 2004). For the second PCR, 1 μl of the PCR product from the initial PCR was used as template. All PCR reactions were performed in 25-μl volumes and we routinely used positive (i.e., DNA from individuals with known malarial infections) and negative (i.e., samples with ddH2O instead of genomic DNA as template) controls to ascertain that the outcome of each PCR run was not affected by contamination (Waldenström et al. 2004). Further, negative infections were confirmed by repeated PCR. The PCR programme, thermal profile, and reagent proportions were as described by Waldenström et al. (2004), with the exception of using a 9-min denaturing at 95°C rather than 3 min at 94°C because we used a hot-start polymerase (EcoStart; Ecogen). All reactions were carried out on a Mastercycler EpgradientS (Eppendorf) thermal cycler. In a previous study including a subset of the samples used here (n = 288), we found that all infections detected by visual screening of blood smears gave positive PCR amplifications while several PCR detected infections could not be determined using visual examinations (Ortego et al. 2007a). As found in several studies, this indicates that the nested PCR protocol is much more sensitive than the traditional microscopy-based examinations of blood smears (Waldenström et al. 2004). Positive or negative second round PCR products (i.e., birds having or not gametocytes or merozoites in their blood stream) were scored by electrophoresis on 2% agarose gels stained with ethidium bromide and determining the presence/absence of a band of the expected size under UV light. PCR products from positive samples were purified using NucleoSpin Extract II (MACHEREY-NAGEL) kits and bidirectionally sequenced on an ABI 310 Genetic Analyser (Applied Biosystems). Sequences were edited and aligned using the program BioEdit (Hall 1999) and are available through GenBank (accession numbers for the three most common lineages analyzed here: RTSR1: AF495568; LK2: EF564175; LK6: EF564179).

Statistical analyses

We analyzed the relationship between reproductive performance and malaria presence using Generalized Linear Mixed Models (GLMMs) implemented with the GLIMIX macro of SAS (SAS Institute 2004). GLMMs allow analyses of data where the response variable is determined by both random and fixed effects. Parameters related with the species reproductive performance were included as response variables in different GLMMs. We considered clutch size (Poisson error and log ling function; males, n = 247; females, n = 280), hatching success (binomial error, logit link function; males, n = 206; females, n = 229), nesting success/failure (binomial error, logit link function; males, n = 240; females, n = 267), and number of fledglings per successful nest that survived at day 30 (Poisson error and log ling function; males, n = 174; females, n = 189) as dependent variables. GLMMs were constructed by fitting presence/absence of the three most common avian malaria lineages (RTSR1, LK2, LK6; Ortego et al. 2007a) parasitizing Lesser Kestrels in the study population as fixed factors together with other terms (covariates: laying date, parental pectoral thickness, parental age; fixed factor: locality) that could potentially influence reproductive performance in this species (e.g., Ortego et al. 2007c). As done in other studies, we developed separate models for males (n = 277) and females (n = 307) because the detrimental consequences of blood parasites on reproductive performance may differ between sexes (Korpimäki et al. 1993; Dawson and Bortolotti 2001). No pair of these independent variables was strongly correlated (all r < 0.35) so we initially included all of them in our analyses (Green 1979). The identities of colonies, cohort, and breeding season were included as random effects to control for the potential non-independence of reproductive parameters within colonies, cohorts, and years in the manner of a randomized complete block design to avoid pseudoreplication (Krackow and Tkadlec 2001).

Initially, each GLMM was constructed with all explanatory terms fitted, including first-order interactions and quadratic effects, to account for potential nonlinear relationships. Final models were selected following a backward procedure, by progressively eliminating non-significant variables. Terms were deleted from the model if their removal caused a non-significant change in deviance (P > 0.05). Thus, the variables retained in the models were those that provided unique explanatory power. The significance of the remaining variables was tested again until no additional variable reached significance. The result is the minimal most adequate model for explaining the variability in the response variable, where only the significant explanatory variables are retained. All tests were performed using the residual degrees of freedom (SAS Institute 2004). Hypotheses were tested using F statistics and all P values refer to two-tailed tests.


Predictably, all studied parameters related with Lesser Kestrel reproductive performance were negatively associated with laying date (Table 1; Perrins 1970). As found in other bird species, clutch size increased and then declined significantly with both male and female age (Table 1; e.g., Kruuk et al. 2002; Reid et al. 2003). Nesting success increased with male age (Table 1). Males parasitized by lineage LK6 raised a lower number of fledglings (Table 1; Fig. 1a). We re-analyzed our data including into the models total prevalence (i.e., all lineages pooled) as a binary variable rather than the presence/absence of each particular avian malaria lineage. Total prevalence did not influence clutch size (males: F1,242 = 1.70, P = 0.193; females: F1,275 = 1.82, P = 0.178), hatching success (males: F1,204 = 1.82, P = 0.179; females: F1,226 = 0.17, P = 0.677), or nesting success (males: F1,236 = 0.08, P = 0.772; females: F1,264 = 0.20, P = 0.652). However, parasitized males raised a lower number of fledglings (F1,171 = 5.40, P = 0.021) and, once again, we found no effect of female parasitic status (F1,186 = 2.75, P = 0.099). Quadratic terms, with the exception of age in clutch size model, and interactions between independent variables were not significant in any analysis (P > 0.1 in all cases).
Table 1

GLMMs for different components of reproductive performance in relation to laying date, age, pectoral thickness, locality, and presence/absence of the three most common malaria lineages (Plasmodium: RTSR1, LK6; Haemoproteus: LK2) parasitizing Lesser Kestrels (Falco naumanni)




Estimate ± SE



Estimate ± SE



Clutch size


1.877 ± 0.157


2.307 ± 0.164


 Laying date

−0.006 ± 0.001

F1,243 = 32.28


−0.008 ± 0.001

F1,276 = 53.67



0.176 ± 0.047

F1,243 = 14.26


0.116 ± 0.034

F1,276 = 11.79



−0.024 ± 0.008

F1,243 = 10.28


−0.013 ± 0.005

F1,276 = 8.35


 Pectoral thickness


F1,242 = 2.03



F1,275 = 0.01




F1,242 = 0.01



F1,275 = 0.64


 Presence of RTSR1


F1,242 = 0.01



F1,275 = 0.01


 Presence of LK6


F1,242 = 0.22



F1,275 = 1.19


 Presence of LK2


F1,242 = 2.23



F1,275 = 0.08


Hatching success



5.439 ± 1.573


 Laying date


F1,204 = 2.69


−0.028 ± 0.012

F1,227 = 5.28




F1,204 = 3.13



F1,226 = 0.40


 Pectoral thickness


F1,204 = 0.18



F1,226 = 2.13




F1,204 = 0.22



F1,226 = 0.01


 Presence of RTSR1


F1,204 = 1.63



F1,226 = 0.01


 Presence of LK6


F1,204 = 1.42



F1,226 = 0.01


 Presence of LK2


F1,204 = 2.39



F1,226 = 0.06


Nesting success/failure


8.032 ± 3.303


9.399 ± 2.432


 Laying date

−0.063 ± 0.023

F1,237 = 7.56


−0.063 ± 0.018

F1,265 = 11.90



0.749 ± 0.352

F1,237 = 4.53



F1,264 = 2.11


 Pectoral thickness


F1,236 = 0.90



F1,264 = 3.38




F1,236 = 0.46



F1,264 = 0.21


 Presence of RTSR1


F1,236 = 0.39



F1,264 = 0.04


 Presence of LK6


F1,236 = 0.82



F1,264 = 0.97


 Presence of LK2


F1,236 = 0.01



F1,264 = 0.01


Number of fledglings per successful nest


2.067 ± 0.483

F1,171 = 7.86


2.537 ± 0.425


 Laying date

−0.010 ± 0.003

F1,171 = 7.86


−0.011 ± 0.003

F1,187 = 11.67


 Presence of LK6

0.321 ± 0.146

F1,171 = 4.83



F1,186 = 2.77




F1,170 = 1.69



F1,186 = 0.01


 Pectoral thickness


F1,170 = 1.61



F1,186 = 0.13




F1,170 = 0.19



F1,186 = 0.88


 Presence of RTSR1


F1,170 = 0.18



F1,186 = 0.16


 Presence of LK2


F1,170 = 3.31



F1,186 = 0.54


Colony identity, cohort, and year were included as random effects in all these analyses. Parameter estimates ± SE were calculated considering a reference value of zero for the parasitized level in the variable “Presence of LK6
Fig. 1

Mean (±SE) number of fledglings per successful nest in relation with a parasitism by lineage LK6 and b total prevalence in male Lesser Kestrels (Falco naumanni). Figures above bars indicate sample size in each group


Here, we analyze the consequences of three different avian malaria lineages on reproductive performance of Lesser Kestrels. Malaria infections in male and female parents had no apparent effects on clutch size, hatching success or nesting success. However, when only successful nests were considered, we found that males parasitized by LK6 raised a lower number of fledglings, suggesting that the level of parental effort by males may be limited by this particular Plasmodium lineage. Such an effect was not detected in females, probably due to a higher proportion of parental investment of males during the chick rearing period as observed by extensive video-tape recording of prey deliveries in several colonies from the study population (G. Calabuig, unpublished data). Similar differences between sexes in the effects of avian malaria parasites or trade-offs between parasitism and reproduction have also been reported in previous studies in relation to differential parental investment (Dawson and Bortolotti 2001; Korpimäki et al. 1993). This result suggests that males suffering from avian malaria may be less efficient foragers, and this could have resulted in a reduced number of raised fledglings. Medication experiments in birds maintaining chronic infections have found that the effects of infection are usually paid for by nestlings, suggesting that the level of parental effort may be limited by malaria parasites as a result of a trade-off between reproductive effort and investment in immune/physiological anti-parasite defence to avoid relapses of chronic infections acquired in previous years (Merino et al. 2000; Tomás et al. 2005). Thus, non-parasitized male Lesser Kestrels could increase the amount of resources devoted to parental care through a reduced allocation to immune response (Merino et al. 2000).

Although we used a relatively large sample size, the effects we found were weak, with a complete absence of negative consequences of the studied avian malaria lineages on most reproductive parameters. The Lesser Kestrel is predominantly infected by only three parasite lineages, a number much smaller than reported for other bird species (e.g., Hellgren 2005; Bensch et al. 2007; Perez-Tris et al. 2007). Further, the low prevalence observed would have led to a low probability of mixed infections (Bensch et al. 2007). Accordingly, no mixed infections have been detected in the study population of Lesser Kestrels (Ortego et al. 2007a), although this may be partially due to the difficulty of detecting concomitant infections because of PCR competition among lineages with different levels of parasitemia (Bensch et al. 2007). In any case, this scarce parasite diversity may have reduced within-host competition between genetically distinct parasites and resulted in low virulence of the studied lineages (Frank 1996). Alternatively, the scarce effects observed may also be a consequence of the correlative nature of this study (Bensch et al. 2007; Ortego et al. 2007d). Thus, the results obtained do not necessarily mean that malaria parasites are harmless for other fitness-related traits (Bensch et al. 2007). In fact, the severest fitness consequences of avian malaria generally occur during the acute infection just after the first exposure to the parasite (Atkinson et al. 2000), whereas individuals surviving the primary infection generally clear the infection or carry chronic stages with supposedly limited consequences on host fitness (Bensch et al. 2007). On the other hand, despite the nested PCR approach being much more efficient than the traditional visual examination of blood smears, parasite prevalence observed in Lesser Kestrels was very low, and this may have reduced the effective power of our statistical analyses in spite of the number of birds screened being relatively high (Ortego et al. 2007d; see also Tella et al. 1996). Further, some birds could be chronically infected without stages of malaria parasites being present in the blood, and these infections may have gone undetected by nested PCR (Jarvi et al. 2002).

Overall, we have found that chronic stages of specific malaria lineages have certain negative consequences on host reproductive performance. This highlights the importance of considering genetic variability among malaria parasites when studying their consequences on natural bird populations. Future studies should consider analyzing intensities of infections of molecularly typed lineages, which could be reliably assessed using real-time PCR techniques (e.g., de Roode et al. 2005). Also, experimental approaches, including medication experiments (Merino et al. 2000; Tomás et al. 2005) or experimental infections (Atkinson et al. 2000; Garvin et al. 2003), could also help to get a better understanding on the consequences of malaria parasites on bird hosts.


Die Folgen chronischer Infektionen mit drei unterschiedlichen Vogelmalaria-Linien für die reproduktive Leistung von Rötelfalken (Falco naumanni)

Wir haben die Folgen chronischer Infektionen mit drei unterschiedlichen Linien der Vogelmalaria, zwei Plasmodium (RTSR1, LK6) und einer Haemoproteus (LK2), für die reproduktive Leistung von Rötelfalken (Falco naumanni) untersucht. Malariainfektionen bei männlichen und weiblichen Eltern hatten keinen Effekt auf Gelegegröße, Schlupferfolg oder Nisterfolg. Wurden jedoch lediglich erfolgreiche Nester berücksichtigt, fanden wir, dass von LK6 parasitierte Männchen weniger flügge Junge aufzogen, was darauf hindeutet, dass das Ausmaß männlicher Brutfürsorge durch diese bestimmte Linie von Plasmodium eingeschränkt sein könnte. Dieser Effekt war bei Weibchen nicht zu beobachten, wahrscheinlich weil Männchen bei dieser Art mehr in die Jungenaufzucht investieren. Insgesamt haben wir herausgefunden, dass chronische Stadien spezifischer Malarialinien gewisse negative Konsequenzen für die reproduktive Leistung des Wirts haben, was hervorhebt, dass es von Bedeutung ist, genetische Unterschiede zwischen Malariaparasiten bei der Untersuchung ihrer Folgen für natürliche Vogelpopulationen in Betracht zu ziehen.


This manuscript was greatly improved by the comments of Robert E. Ricklefs and an anonymous reviewer. This work received financial support from the projects: CGL2005-05611-C02-02/BOS (Ministerio de Educación Ciencia) and PAI05-053 (Junta de Comunidades de Castilla-La Mancha). During this work J.O and G.C. were supported by predoctoral fellowships from the Junta de Comunidades de Castilla-La Mancha and the European Social Fund. We performed all the laboratory work at the Laboratory of Genetics of the IREC and sequencing was performed by the Centro de Investigaciones Biológicas (CSIC) of Madrid. We manipulated and banded Lesser Kestrels under license from the Spanish institutional authorities (Environmental Agency of the Community of Castilla-La Mancha and the Ringing Office of the Ministry of Environment) and we followed general ethical guidelines for animal welfare and nature conservation.

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© Dt. Ornithologen-Gesellschaft e.V. 2008