ORIGINAL ARTICLE

High prevalence of haemosporidians in Reed WarblerAcrocephalus scirpaceus and Sedge Warbler Acrocephalusschoenobaenus in Spain

Monica Fernandez Æ Ma. Angeles Rojo ÆPatricia Casanueva Æ Silvia Carrion ÆMa. Angeles Hernandez Æ Francisco Campos

Received: 5 September 2008 / Revised: 9 February 2009 / Accepted: 28 April 2009 / Published online: 21 May 2009

� Dt. Ornithologen-Gesellschaft e.V. 2009

Abstract Apicomplexan blood parasites (genera Haemo-

proteus, Plasmodium and Leucocytozoon) prevalence in two

related species (Reed Warbler Acrocephalus scirpaceus and

Sedge Warbler A. schoenobaenus) was studied in 2006 at the

Natural Reserve of Castronuno-Vega del Duero, Western

Spain, a stopover area during the autumn migration. A

fragment of the mitochondrial cytochrome b gene of the

parasites was amplified, using a nested PCR assay, from

avian blood samples. High prevalence of malaria parasites

was found in both species, 84.6% in Reed Warbler and 71.8%

in Sedge Warbler, and the degree of infection reach 100% of

the population that breed at the Reserve, suggesting good

conditions for the development of dipteran vectors in this

area. By sequencing 464 nucleotides of the obtained frag-

ments, we found four different mitochondrial haplotypes of

Haemoproteus or Plasmodium in the two species analysed.

Leucocytozoon infection was not detected, in contrast to the

high prevalence of this parasite in other avian species in

Spain, probably because the water course studied is not an

adequate habitat for its vectors.

Keywords Acrocephalus � Avian malaria �Haemosporidians � Spain

Introduction

Species of the apicomplexans, Haemoproteus, Plasmodium

and Leucocytozoon comprise a diverse group of vector-

transmitted parasites that infect red blood cells in their

vertebrate host and also white blood cells in the case of

Leucocytozoon (Atkinson and Van Riper 1991; Valkiunas

1993). These parasite genera are genetically closely related

and all three are referred to as avian malaria (Perkins and

Schall 2002; Ricklefts et al. 2004; Perez-Tris et al. 2005).

However, life cycle, vector species and epidemiology of

parasites from different families are distinct; thus, the tra-

ditional view accepts only Plasmodium species as being the

true malaria parasite (Valkiunas 2005).

A blood infection rate of 61% was recorded by Merino

et al. (1997) in 16 different species of passerine birds in the

Iberian Peninsula, with Leucocytozoon as the most frequent

parasite (39% incidence, as opposed to 17 and 1% for

Haemoproteus and Plasmodium, respectively). Munoz et al.

(1999) analysed samples of strigiformes and falconiformes,

in which Leucocytozoon was also the most common parasite,

the level of infection from hematozoa varying from 46.3%

(falconiformes) to 30.5% (strigiformes). Similarly, Tella

et al. (1999) reported levels of infection (predominantly

Haemoproteus, followed by Leucocytozoon) which did not

exceed 40% in any of the analysed species.

In other areas of Europe, Scheuerlein and Ricklefts

(2004) indicated that Haemoproteus was the most widely

found genus in several species of passerines (mean infec-

tion rate 26%), concurring with the fact that this parasite

causes mild effects in the host (Atkinson and Van Riper

1991), while Plasmodium appears to be the most invasive

hemoparasite for birds (Atkinson et al. 2000).

Through nested PCRs for simultaneous diagnosis of

Leucocytozoon, Plasmodium and Haemoproteus, Hellgren

Communicated by C. G. Guglielmo.

M. Fernandez (&) � Ma. A. Rojo � P. Casanueva � S. Carrion �F. Campos

European University Miguel de Cervantes,

Padre Julio Chevalier 2, 47012 Valladolid, Spain

e-mail: mfernandez@uemc.es

Ma. A. Hernandez

Department of Zoology and Ecology, School of Sciences,

University of Navarra, 31080 Pamplona, Spain

123

J Ornithol (2010) 151:27–32

DOI 10.1007/s10336-009-0417-z

et al. (2004) obtained a higher degree of infection in the

Bluethroat Luscinia svecica from Sweden (59%), with

Leucocytozoon still the most widely represented parasite

(48% of birds analysed), followed by Plasmodium (24%)

and Haemoproteus (1.2%).

With regard to the genus Acrocephalus, Buchanan et al.

(1999), Bensch et al. (2000) and Shurulinkov and Chakarov

(2006) have contributed data on the percentage of infected

Reed Warblers from Africa, Asia and Europe. Bensch et al.

(2000) analysed only five samples of Reed Warbler from

the Iberian Peninsula, and Shurulinkov and Chakarov

(2006) obtained their results through blood smear obser-

vation; this morphological technique provides similar

results in hemoparasite detection and prevalence studies

than molecular methods (Valkiunas et al. 2008). However,

PCR and sequencing protocols are needed for the detection

of parasites belonging to different haplotypes (Valkiunas

et al. 2007; Krone et al. 2008).

Using molecular techniques, this study focused on the

presence of Leucocytozoon, Plasmodium and Haemopro-

teus, and their different haplotypes, in two species of the

genus Acrocephalus (Reed Warbler A. scirpaceus and

Sedge Warbler A. schoenobaenus) from Spain, analysing

the degree of infection according to bird age and sex, and

distinguishing between nesting individuals and those

stopping over.

Methods

Study area

The study was run in the Natural Reserve of Castronuno in

Midwestern Spain (41�240N, 05�150W, 660 m asl, mean

annual temperature 12�C). A small reservoir (6 hm3) built

on the river Duero has favoured the growth of an extensive

giant reed bed (Phragmites australis) which houses

an abundant nesting population of Reed Warblers. In July

and August, this location is a stopover area for Reed

Warblers and Sedge Warblers; however, the latter do not

breed there.

Birds were captured in June, August and September

2006 with mist nets placed from sunrise until 4 h later. All

were classified by age following the Svensson (1996) cri-

teria into adults (at least 1 year old) or yearlings. Following

the breeding timing observed by Bermejo (2004) for Reed

Warblers in Central Spain, those captured in June were

considered as belonging to the breeding population, and

those captured in August and September were considered

as on migratory passage.

A blood sample (\0.1 ml) was obtained by brachial vein

puncture from birds selected at random and stored on

FTA� cards.

DNA extraction

Total DNA from FTA� cards blood samples was extracted

with ammonium acetate (Gutierrez-Corchero et al. 2002).

A piece of the dried blood sample (approximately 2 mm2)

was cut away using a sterile cutter and transferred to a

microcentrifuge tube and incubated in 250 ll SET buffer

(0.15 M NaCl, 0.05 M Tris pH 7.5, 0.001 M EDTA) at 4�C

during 5 h. The sample was then treated with 7 ll of SDS

20% and 5 ll of proteinase K (10 mg/ml) at 55�C o/n,

250 ll of amonium acetate 4 M was added to the mixture

and left at room temperature for 30 min after which the

suspension was centrifuged for 10 min at 13,500 rpm. The

supernatant was transferred to a new microcentrifuge tube

and DNA was precipitated by adding 1 ml of absolute

ethanol. After centrifugation, the air-dried DNA pellet was

resuspended in 25 ll of MQ water.

Amplification of the avian CHD-Z y CHD-W genes

To determine the gender of the birds a fragment of CHD-Z

and CHD-W genes was amplified by polymerase chain

reaction (PCR), using synthetic oligonucleotides P8 and

P2 (Sigma-Aldrich�) designed by Griffiths et al. (1998).

Reaction conditions and amplification programme used

were those described by Gutierrez-Corchero et al. (2002) in

a Techne TC 412 thermocycler. After amplification, two

bands of an estimated size of 350 and 380 bp for females

and a single band of approximately 350 bp for males were

obtained and visualised in 2.5% agarose gel.

Amplification of the parasite mitochondrial cyt b gene

For the amplification of a small part of the mitochondrial

gene cyt b, a nested chain reaction (PCR) protocol was

used with HaemFNI/HaemR3N//HaemF/HaemR2//HaemFL/

HaemR3L (Sigma-Aldrich�) primers as described in Hellgren

et al. (2004). The method obtains a 478-bp fragment

(excluding the primers) of the cyt b for Leucocytozoon and

480-bp fragment for Haemoproteus and Plasmodium. To

double-check the success of the reactions, 5 ll of the final

PCRs were used for running on a 1.5% agarose gel.

This protocol makes it possible to differentiate between

individuals infected by Leucocytozoon and those infected

by Plasmodium or Haemoproteus. However, differentiation

of the infection between these last two genera requires

further sequencing of the amplified cyt b fragment.

Sequencing and analysis of molecular data

Some samples showing positive amplification were random

selected for sequencing using procedures as described

28 J Ornithol (2010) 151:27–32

123

by Bensch et al. (2000). The fragments (Haemoproteus–

Plasmodium positives samples) were sequenced directly

from 50end with HaemF primer using dye terminator cyclic

sequencing and loaded on an ABI PRISMTM

310 (Perkin

Elmer).

The 464-bp DNA sequences were edited and aligned

using the Lasergene� 7.1 software by DNASTAR and

analysed with the NCBI worldwide web blast server (www.

ncbi.nlm.nih.gov/blast).

The haplotypes obtained have been deposited at the

Genbank International Nucleotide Sequence Database with

accession numbers EU438120, EU438121, EU438122,

EU438123, EU709729 y EU709730.

For statistical analysis of the data, the v2 test was used

with Yates correction when the frequency of observations

was \10, and Fischer’s exact test when the expected fre-

quency was \5.

Results

A total of 149 Reed Warblers were analysed, out of which

126 (84.6%) showed infection, and 39 Sedge Warblers, of

which 28 (71.8%) were infected, with no significant dif-

ferences between the two values (v12 = 3.40, P [ 0.05).

In the Reed Warbler, the percentage of infected birds

varied significantly with the season of the year (greater in

the breeding season than during autumn migration) and

with sex (more infected males than females) but not with

age group (Table 1). In contrast, the Sedge Warbler did not

show any significant differences between percentages of

infected birds when analysed by sex and age group

(Table 1).

All infected Reed Warblers (n = 126) and Sedge War-

bler (n = 28) were negative for Leucocytozoon and

positive for Plasmodium–Haemoproteus. To distinguish

between the infection in these last two genera, some

positive samples of both species were sequenced (Table 2).

Samples sequenced showing multiple infections (7.57%)

have been discarded and kept for further studies. In the

Reed Warbler, 97.67% of the samples sequenced belonged

to three new Haemoproteus haplotypes (Table 2): MAPH1

(acc. no. EU438120, 2.32% of samples), MAPH2 (acc. no.

EU438122, 76.74%) and MAPH3 (acc. no. EU438123,

18.6%) and only one sample belonged to Plasmodium (new

haplotype MAPP1, acc. no. EU438121, 2.32%). In the

Sedge Warbler, all the sequenced samples revealed the

presence of Haemoproteus (Table 2), and haplotypes

obtained were MAPH2 (acc. no. EU709729, 94.5% of

samples) and MAPH3 (acc. no. EU709730, 5.5%), both of

them belonging to the two most frequent representations of

haplotypes for Reed Warblers.

Discussion

All the Reed Warblers analysed in the area of study during

the breeding season and 78.3% of those which were on

migratory passage showed infection by haematozoa. These

percentages of infected birds are the highest recorded up to

now in the genus Acrocephalus. It is well known that in

passerines the prevalence of hemoparasites may vary

widely between geographical regions (e.g. Merila et al.

1995; Bensch and Akesson 2003) and that the abundance

of insects transmitting Plasmodium and Haemoproteus

depends greatly on local climatic conditions (Turell 1989;

Rueda et al. 1990).

The study area was located within the Mediterranean

region of the Iberian Peninsula where the temperature has

experienced an increase in recent decades (Moberg and

Jones 2005). More specifically, in 2006, the mean tem-

perature in the study area during the months of June–

September was 1.2�C higher than the temperature recorded

in the 1970–2005 period. This suggests that, during the

sampling year, the abundance of vectors in this area may

have been significant, and consequently the probability of a

bird becoming infected was also high. The stopover time of

Reed Warblers in the Reserve during migratory flight was

not known; however, given the environmental conditions,

it is probable that many were rapidly infected.

In the two species analysed, the degree of infection did

not vary with age. This result is contrary to what has been

found in other species of passerines, where infection was

higher in adults (Allander and Bennett 1994). In Reed

Warblers (but not in Sedge Warblers, as was also shown by

Buchanan et al. 1999), the percentage of infected birds

varied with sex: higher in males than in females. In some

passerines species, males showed greater prevalence of

Table 1 Percentage of Reed Warblers Acrocephalus scirpaceus and

Sedge Warblers A. schoenobaenus infected and non-infected by

haematozoa in the study according to the season (breeding or

migration), sex (males, females) and age (yearlings, adults)

Species Parameter n Infected Non-

infected

v2 test

Reed Warbler Breeding 31 100 0 5.73, P \ 0.005

Migration 118 80.5 19.5

Males 94 91.4 8.6 7.97, P \ 0.005

Females 55 72.7 27.3

Yearlings 81 83.9 16.1 0.04 n.s.

Adults 68 85.2 14.8

Sedge Warbler Males 22 68.1 31.9 0.33 n.s.

Females 17 76.4 23.6

Yearlings 29 65.5 34.5 1.16 n.s.

Adults 19 90.0 10.0

J Ornithol (2010) 151:27–32 29

123

hemoparasites, when both breeding effort (Norris et al.

1994) and testosterone levels increased (Deviche and Parris

2006). It is possible that breeding or passing males ana-

lysed in this study suffered a high degree of wear and tear

in the breeding period immediately before capture and

therefore their probability of being infected was greater.

In the samples obtained, Haemoproteus was the most

abundant parasite. Within the genus Acrocephalus,

Buchanan et al. (1999) recorded Haemoproteus in 2.4–

15.6% breeding Sedge Warblers from the UK, and Bensch

et al. (2000) recorded it in the Basra Reed Warbler

A. griseldis from Kenya (40% of samples). Shurulinkov and

Chakarov (2006) recorded Haemoproteus in Reed Warbler

(maximum 42.9% of samples) and Great Reed Warbler

A. arundinaceus (maximum 23.9%) from Bulgaria. Simi-

larly, this study indicates the lack of presence of Plasmodium

in Sedge Warbler and very low prevalence in Reed Warbler

(Table 2). This had also been recorded earlier in species of

Acrocephalus, such as Sedge Warbler (Buchanan et al.

1999; Waldenstrom et al. 2002; Beadell et al. 2006) and

Great Reed Warbler (Shurulinkov and Chakarov 2006).

The 464-bp DNA fragments haplotypes MAPH2 and

MAPH3 found in Reed Warbler and Sedge Warbler had,

respectively, 99% identity to Haemoproteus payevskyi

RW1 (DQ630009) from Reed Warbler from Lithuania

(Hellgren et al. 2007) and 99% identity to Haemoproteus

payevskyi GRW1 (AY560361) from Great Reed Warbler

from Sweden (Bensch et al. 2004). The third Haemopro-

teus, haplotype MAPH1, found only in Reed Warbler,

had 98% identity to Hameproteus beloposkyi HIICT1

(DQ630006) from Icterine Warbler Hippolais icterina from

Sweden (Hellgren et al. 2007). Finally, the only Plasmo-

dium registered showed 99% identity to Plasmodium

elongatum p52 (DQ659588) from Great Blue Heron Ardea

herodias from the USA (Beadell et al. 2006).

The idea that related species share the same or close

Haemoproteus or Plasmodium has been previously devel-

oped (Waldenstrom et al. 2002; Szymansky and Lovette

2005). The fact that the same species of parasite has several

different hosts is related to frequent changes in its viru-

lence, and these hemoparasites play a complex role in the

evolutionary history of the host (Toft and Karter 1990).

Bensch et al. (2000) showed the presence of single hap-

lotypes of the gene cyt b in each species, with the exception

of a haplotype of Haemoproteus majoris, which is repeated

in two related species (Blue Tit Cyanistes caeruleus and

Great Tit Parus major). Waldenstrom et al. (2002) showed

that 44% of the lineages of haemosporidians analysed were

repeated in more than one host species from Europe or

Africa, a lineage of Plasmodium even infecting birds from

different families. Similarly, Reullier et al. (2006) con-

firmed that two haplotypes of Haemoproteus were repeated

in two species of birds of the genus Hippolais. Hellgren

et al. (2007) have suggested that parasites belonging to the

genus Haemoproteus or Leucocytozoon were restricted to

avian fauna resident in an area and which rarely modified

their area of transmission, whereas the lineages of the

genus Plasmodium spread more easily, probably due to the

tendency to infect migratory species.

Our data show the absence of Leucocytozoon in Reed

Warblers and Sedge Warblers analysed. Earlier Shurulin-

kov and Chakarov (2006) detected the presence of a single

example of Reed Warbler and Great Reed Warbler infected

by Leucocytozoon fringillinarum. This absence may be due

to many different factors such as, for example, the high

virulence of Leucocytozoon (Cordero del Campillo 1999),

which may cause the death of birds before they are cap-

tured; the scarcity or absence of vectors (Simulium sp.) in

the study area, since the water there is reservoir water and

unfavourable to the presence of these insects (Valkiunas

et al. 2005), and feeding habits which are so different in

vectors and birds that they do not allow space–time syn-

chronisation between each other (Greiner 1991; Atkinson

1999). Further studies are therefore needed to explain this

absence.

Zusammenfassung

Starke Verbreitung von Haemosporidien bei

Teichrohrsangern (Acrocephalus scirpaceus) und

Schilfrohrsangern (Acrocephalus schoenobaenus)

in Spanien

Die Verbreitung von Blutparasiten der Apicomplexa (Gen-

era Haemoproteus, Plasmodium und Leucocztozoon) wurde

in zwei verwandten Arten (Teich- und Schilfrohrsanger) im

Jahr 2006 im Naturschutzgebiet von Castronuno-Vega del

Table 2 Number of Reed Warblers and Sedge Warblers infected by Plasmodium (MAPP1) and Haemoproteus (MAPH1, MAPH2 and MAPH3)

during the breeding season (June) and autumn migration (August–September)

Species Period Infected Sequenced MAPH1 MAPH2 MAPH3 MAPP1

Reed Warbler Breeding 31 24 0 15 8 1

Migration 95 19 1 18 0 0

Sedge Warbler Migration 28 18 0 17 1 0

30 J Ornithol (2010) 151:27–32

123

Duero in Westspanien untersucht, das ein Rastplatz wahrend

des Herbstzugs ist. Ein Fragment des mitochondrialen

Cytochrom B Gens des Parasiten wurde mit einer nested

PCR aus Blutproben der Vogel amplifiziert. Eine starke

Verbreitung von Malaria-Parasiten wurde bei beiden Arten

gefunden, 84.6% bei Teichrohrsangern und 71.8% bei

Schilfrohrsangern; die Infektionsrate in diesem Gebiet

erreicht 100%, was fur gute Bedingungen fur die Entwickl-

ungen des dipterischen Vektors spricht. Durch die Sequen-

zierung von 464 Nukleotiden der amplifizierten Fragmente

fanden wir vier unterschiedliche mitochondriale Haplotypen

von Haemoproteus oder Plasmodium bei den untersuchten

Arten. Eine Infektion mit Lezcocytozoon wurde im Gegen-

satz zu ihrem starken Auftreten bei anderen Vogelarten in

Spanien nicht gefunden, wahrscheinlich weil die Gewasser

im Untersuchungsgebiet ihrem Vektor kein adaquates

Habitat bieten.

Acknowledgments This study was financed by Obra Social of Caja

Espana. Our thanks to Alberto Galan and Cristina Miranda for their

enthusiastic and efficient assistance on the field and to Ana Amezcua,

Barbara Gutierrez and Yolanda Fernandez for their lab and statistics

studies. The Castilla and Leon Regional Government provided the

official permits for bird capturing. The Centro Meteorologico of

Castilla and Leon provided the meteorological data. We thank two

anonymous referees for helpful comments on the manuscript.

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