Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

High Prevalence and Lineage Diversity of Avian Malaria in Wild Populations of Great Tits (Parus major) and Mosquitoes (Culex pipiens)

  • Olivier Glaizot ,

    olivier.glaizot@unil.ch

    Affiliations Museum of Zoology, Lausanne, Switzerland, Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

  • Luca Fumagalli,

    Affiliations Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland, Laboratory for Conservation Biology, Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

  • Katia Iritano,

    Affiliation Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

  • Fabrice Lalubin,

    Affiliation Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

  • Juan Van Rooyen,

    Affiliation Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

  • Philippe Christe

    Affiliation Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland

High Prevalence and Lineage Diversity of Avian Malaria in Wild Populations of Great Tits (Parus major) and Mosquitoes (Culex pipiens)

  • Olivier Glaizot, 
  • Luca Fumagalli, 
  • Katia Iritano, 
  • Fabrice Lalubin, 
  • Juan Van Rooyen, 
  • Philippe Christe
PLOS
x

Abstract

Avian malaria studies have taken a prominent place in different aspects of evolutionary ecology. Despite a recent interest in the role of vectors within the complex interaction system of the malaria parasite, they have largely been ignored in most epidemiological studies. Epidemiology of the disease is however strongly related to the vector's ecology and behaviour, and there is a need for basic investigations to obtain a better picture of the natural associations between Plasmodium lineages, vector species and bird hosts.

The aim of the present study was to identify the mosquito species involved in the transmission of the haemosporidian parasites Plasmodium spp. in two wild populations of breeding great tits (Parus major) in western Switzerland. Additionally, we compared Plasmodium lineages, based on mitochondrial DNA cytochrome b sequences, between the vertebrate and dipteran hosts, and evaluated the prevalence of the parasite in the mosquito populations. Plasmodium spp. were detected in Culex pipiens only, with an overall 6.6% prevalence. Among the six cytochrome b lineages of Plasmodium identified in the mosquitoes, three were also present in great tits. The results provide evidence for the first time that C. pipiens can act as a natural vector of avian malaria in Europe and yield baseline data for future research on the epidemiology of avian malaria in European countries.

Introduction

Transmission of avian malaria is highly dependent on the distribution, competence and vectorial capacity of vectors. Despite a recent increase in interest toward dipteran insects as major actors in avian malaria systems [1][4], there is a lack of general knowledge of the role of the vectors in this mosquito-borne disease. Most of the information on the vectorial competence of diverse mosquito species has been reviewed by Huff [5] but natural avian malaria vectors have been mostly ignored until very recently [6][8]. Although many species seem to be competent according to laboratory experiments [6], only a few recent studies have identified natural vectors responsible for avian malaria transmission in different parts of the world, with the notable exception of Europe [1], [2], [9][16].

Molecular studies on avian malaria have permitted the investigation of questions concerning phylogeny, phylogeography and life-history evolution of Plasmodium parasites [17][21]. However, the epidemiological success of Plasmodium strongly depends on its life cycle within the mosquito and in the vector's ability to find suitable hosts for taking a blood meal. Furthermore, asexual reproduction of Plasmodium occurs in vertebrate hosts whereas sexual reproduction takes place in the mosquito, making the vector the definitive host [22]. Mosquitoes are thus expected to play a major role in both avian malaria parasites' evolution and disease transmission [23], [24]. It is therefore of prime interest to deepen the understanding of the interactions between the different actors of this complex host-parasite system under wild conditions, the first step being the identification of the vector in such wild populations.

The aims of the present study were to identify the natural vector of avian malaria in two wild populations of great tits (Parus major) in Switzerland, where the prevalence of infection has been shown to be high [20], and to identify and compare malaria lineage diversity among the bird hosts and the mosquito vectors by means of mitochondrial cytochrome b gene sequencing.

Materials and Methods

Ethical notes

All necessary permits were obtained for the described field studies and the study was approved by the «Conservation de la Faune du Canton de Vaud». No other specific permissions were required for this study and study sites were non-protected, public properties. This study did not involved endangered or protected species. All birds were treated in accordance with the Cantonal Veterinary Authorities of the Canton de Vaud, Switzerland, authorization 1730. Captures of birds were made under license number F044-0799 of the Swiss Federal Office for the Environment.

Study sites

The study was carried out in 2006 and 2007 in two populations of great tits (Parus major) breeding in nestboxes. The first population was located in the forest of Dorigny, on the campus of the University of Lausanne (46°31′N; 6°34′E; alt. 400 m). This forest is mainly composed of beech (Fagus sylvatica) and a mixture of large dominant deciduous species like oak (Quercus) and hornbeam (Carpinus). The second study area was located in the forest of Monods (46°34′N; 6°24′E; alt. 680 m). This mixed forest is composed predominantly of beech and spruce (Picea abies) and surrounded by an adler (Alnus glutinosa) forest flooded with stagnant water.

Sampling of mosquitoes

Mosquitoes were sampled from July to September, using both BG-Sentinel traps (BioGents GmbH, Germany) and gravid mosquito traps (Bioquip, California). The first device, using CO2 (dry ice) as a lure, was used to collect host-seeking mosquitoes whereas gravid mosquito traps were intended to capture fed females just before or after oviposition. As egg laying usually takes place five (up to ten) days after blood feeding [25], complete digestion should have occurred at the time of oviposition ensuring absence of remnant bird blood in the digestive tract. As a precaution, however, female mosquitoes were kept alive for 48 hours after trapping before being killed. Mosquito species were identified by morphology in the laboratory.

Sampling of birds

Adult great tits were trapped in their nestboxes during the breeding season by using door traps mounted inside the nestboxes when their nestlings were fourteen days old. A blood sample (20 µl) was then taken by brachial venapuncture and stored at −20°C until molecular assessment of malarial lineages.

Molecular analyses

DNA was extracted from thoraxes of unfed mosquitoes and from adult bird blood samples using the DNeasy tissue extraction kit (QIAGEN) according to the manufacturer's protocol, and was resuspended in 200 µl TE buffer.

Parasite lineages were detected by using a nested PCR method developed by Waldenström et al. [26] from the original protocol [17], amplifying a portion of the mitochondrial DNA (mtDNA) cytochrome b gene. Full details of PCR and sequencing conditions are described in Christe et al. [20]. Sequences were edited and aligned using the program SEQUENCHER 3.0 (Gene Codes, Ann Arbor, Michigan) with additional manual editing. Mitochondrial DNA lineages were identified by comparison with published sequences available on GenBank, and named according to the MalAvi database [27]. Sequences overlapping with less than the 478-bp reference sequence of the MalAvi database were assigned to a precise haplotype name only if they were identical to a reference sequence present in the database. For linkage between mtDNA lineages and morphospecies, we referred to studies combining molecular and morphological approaches [28][30] as well as publications matching cytochrome b haplotypes to a sequence in GenBank associated with a particular morphotype. Sequences were deposited in GenBank (Accession Nos. JQ778276–JQ778282).

Results

Six genera of mosquitoes representing a total of 15 species were captured from the study populations. Among them, nine species (four genera) are mainly or potentially ornithophilous and were analyzed for the presence of Plasmodium and Haemoproteus parasites (Table 1). Only Culex pipiens individuals were found to be positive for avian malaria with an overall prevalence of 6.6% (n = 394).

thumbnail
Table 1. Analyses of ornithophilous mosquito species per study location.

https://doi.org/10.1371/journal.pone.0034964.t001

All infected C. pipiens originated from the Dorigny forest (26 out of 330 individuals collected, 7.9%), whereas none of the 64 collected in the Monods forest were found to be infected. Proportions of infected birds at both locations were comparable, with 91% of infected birds in the Monods forest and 98% in Dorigny.

Six different Plasmodium lineages were identified among the 26 infected Culex pipiens, and four among the 54 great tits analysed (Table 2). One lineage of Haemoproteus, known to be transmitted by non-mosquito vectors, was found in a great tit individual.

thumbnail
Table 2. Plasmodium and Haemoproteus lineages detected by sequencing a portion of the mtDNA cyt b gene (433–478 bp) in 54 Parus major and 26 Culex pipiens individuals.

https://doi.org/10.1371/journal.pone.0034964.t002

Discussion

The relatively high prevalence of Plasmodium spp. found in females Culex pipiens strongly suggests that this species acts as a vector of avian malaria in one of our study populations. C. pipiens was infected with six Plasmodium lineages, among which three were also identified in great tits. Haemoproteus spp., transmitted by biting midges of the genus Culicoides [31], was present in one great tit individual.

Many species of Culex have been described as vectors of avian malaria parasites and their role as competent vectors in experimental infections has been proven [32]. C. pipiens, or the two subspecies C. p. pipiens and C. p. pallens, have been recently reported to be the natural vector of avian malaria in Japan [1], [10][14] and North America [2]. Other species of the genus Culex have been described as vectors of avian Plasmodium: C. restuans in North America [2], [16], C. (Melanoconion) ocossa in Panama [8], C. saltanensis in Brazil [33], C. sasai in Japan [14], C. quinquefasciatus in Japan [12] Hawai'i [34] and Mexico [15], C. neavei, C. perfidiosus, C. poicilipes and C. guiarti in Cameroon [35] and finally C. sitiens and C. annulirostris in the southwest Pacific islands [9]. The results of the present study suggest therefore for the first time the role of Culex pipiens in the transmission of avian malaria in the European continent.

None of the mosquitoes captured in Monods were positive for malaria, although prevalence in great tits was 91%. This could be due to our low sample size of parous females and/or to other vector species involved in the transmission of Plasmodium present in this particular site. Culiseta morsitans has been shown to be the vector of Plasmodium circumflexum in waterfowl in New Brunswick [36], whereas Kimura et al. [2] did not find any Plasmodium in 176 Culiseta melanura in Ithaca (USA). Furthermore, C. morsitans was also described as competent vector of P. polare [6], which presents an overall prevalence of 24% and 19.2% among great tits and infected C. pipiens, respectively. The low number of Culiseta morsitans (n = 31) sampled in the present study did not permit the exclusion of this species as potential vector and ongoing studies should elucidate this issue.

Mosquito samplings may not reflect the relative abundances of species in the study sites. First, the type of traps or attractant used may be specific to one or several species [37]. Secondly, capture success of a given species may depend on the trap location itself. A recent study found a higher capture rate for C. pipiens at ground compared to canopy level, whereas biting midges (Culicoides spp.) (Diptera: Ceratopogonidae), known to be involved in the transmission of Haemoproteus, showed the opposite pattern [38]. Similarly, the opportunistic Anopheles plumbeus showed a spatial preference for canopy level [38]. In the present study, gravid mosquito traps were placed at ground level whereas sentinel traps caught mosquitoes approximately 3 meters from the ground. An ongoing study on overall mosquito diversities (Glaizot, unpublished) revealed that C. pipiens was the most caught mosquito species in the Dorigny population (723/953 = 75.9%) whereas it represents 12.1% of the captures in Monods (150/1237). In this latter population, the other main ornithophilic species (Culiseta alaskaensis and C. morsitans) were captured at a rate of 7.1% and 5.2%, respectively. A bias in the trapping method cannot be excluded and further studies should also concentrated on mosquito trapping at the canopy level. Apart strict ornithophilic species listed in Table 1, some mammophilic and opportunistic species are abundant in the study populations. For example, Anopheles plumbeus is widespread in the Dorigny population (136/953 = 14.3%) whereas the mammophilic Ochlerotatus rusticus (579/1237 = 46.8%) and Ochlerotatus annulipes/riparius/cantans (283/1237 = 22.9%) are the most abundant species in Monods.

In the present study, parasite DNA was extracted from dissected mosquito thoraxes. One potential problem to this approach is that sporozoites, the ultimate parasite life stage before transmission to birds, can occur in the haemocoel during their travel from the midgut to the salivary glands but never reach and fully develop in the salivary glands. Consequently, the amplified parasite DNA from mosquito thoraxes may come from non-infectious parasite stages, and therefore non competent mosquitoes [39]. Experimental infections and/or microscopic detection of both oocysts in the midgut and sporozoite in the salivary glands would fully confirm the role of C. pipiens as a natural vector in the study populations. However, the absence of Plasmodium DNA in the other mosquito species captured and the presence of three lineages found in both birds and mosquitoes strongly suggests that C. pipiens is an active actor for the transmission of Plasmodium spp to Parus major. Moreover, an ongoing study (Lalubin et al., unpublished), which combines molecular detection methods with morphological examination of mosquito midguts and salivary glands, confirms the role of C. pipiens in the transmission cycle of avian malaria.

P. relictum (lineages SGS1, GRW11 and P5) was the most abundant haemosporidian parasite found in the great tit populations and one of the main morphospecies responsible for infection in C. pipiens at Dorigny, a result similar to those reported for Japan [1]. In laboratory conditions, P. relictum develops in more than 20 mosquito species including C. pipiens [32] belonging to six genera and has a large bird host range including more than 300 species from different orders and a worldwide distribution [6]. P. polare (SW2) was well represented both in the bird and mosquito samples. According to Valkiunas [6], this parasite has been recorded among 22 bird species in all zoogeographical regions except Antartica and Australia. However, to our knowledge, the present study is the first to report its presence in C. pipiens. Another common lineage, TURDUS 1, assigned to the morphospecies P. circumflexum, has also been found both in great tits and, for the first time, in C. pipiens. This morphospecies has been recorded in over 100 bird species across all regions except the Antartic [6]. Finally, lineage SYAT05, assigned to Plasmodium vaughani, has been found in more than 200 Passeriformes as well as other bird orders [6]. C. pipiens and C. restuans have been reported as vectors for this lineage [2].

Knowledge on temporal and spatial patterns of parasite transmission is essential to understand the selective pressure imposed by parasites on their hosts. For the first time described as a natural vector of avian malaria in Europe, Culex pipiens seems to play a major role in the transmission of a high range of Plasmodium morphospecies. The higher diversity of lineages of Plasmodium found in C. pipiens compared to those found in great tits also suggests that C. pipiens is a natural avian malarial vector for several other bird species. Furthermore, the presence of some of these Plasmodium lineages in bird species sampled in Africa [40] suggests that in these migrating species, infection may also occur in Europe.

Most of the malarial studies to date have used non-natural systems (i.e. hosts and parasites that would not encounter one another in nature). Using vertebrate/mosquito/haemosporidian model systems from natural populations of wild species [41][43] may provide powerful insights into the complex interactions and constraints affecting the relationship between hosts and parasites.

Acknowledgments

The late Katia Iritano initiated this study during her Master thesis. We thank Staffan Bensch and Patrick Durand for the appreciated advises for the lab work, Nadia Bruyndonckx and Anne-Lyse Ducrest for laboratory assistance, Laurent Brügger for field work, Geneviève L'Eplattenier for help with mosquito dissections, the two anonymous reviewers and the editor for helpful comments.

Author Contributions

Conceived and designed the experiments: OG PC. Performed the experiments: KI PC OG LF FL JR. Analyzed the data: KI LF OG PC. Wrote the paper: OG PC LF.

References

  1. 1. Kim KS, Tsuda Y (2010) Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community. Molecular Ecology 19: 5545–5554.KS KimY. Tsuda2010Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community.Molecular Ecology1955455554
  2. 2. Kimura M, Darbro JM, Harrington LC (2010) Avian Malaria Parasites Share Congeneric Mosquito Vectors. Journal of Parasitology 96: 144–151.M. KimuraJM DarbroLC Harrington2010Avian Malaria Parasites Share Congeneric Mosquito Vectors.Journal of Parasitology96144151
  3. 3. Njabo KY, Cornel AJ, Sehgal RNM, Loiseau C, Buermann W, et al. (2009) Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa. Malaria Journal 8: 193.KY NjaboAJ CornelRNM SehgalC. LoiseauW. Buermann2009Coquillettidia (Culicidae, Diptera) mosquitoes are natural vectors of avian malaria in Africa.Malaria Journal8193
  4. 4. LaPointe DA, Goff ML, Atkinson CT (2010) Thermal Constraints to the Sporogonic Development and Altitudinal Distribution of Avian Malaria Plasmodium Relictum in Hawai'i. Journal of Parasitology 96: 318–324.DA LaPointeML GoffCT Atkinson2010Thermal Constraints to the Sporogonic Development and Altitudinal Distribution of Avian Malaria Plasmodium Relictum in Hawai'i.Journal of Parasitology96318324
  5. 5. Huff CG (1965) Susceptibility of mosquitoes to avian malaria. Experimental Parasitology 16: 107–132.CG Huff1965Susceptibility of mosquitoes to avian malaria.Experimental Parasitology16107132
  6. 6. Valkiunas G (2005) Avian malaria parasites and other haemosporidia. Boca Raton: CRC Press. G. Valkiunas2005Avian malaria parasites and other haemosporidiaBoca RatonCRC Press
  7. 7. Massey B, Gleeson DM, Slaney D, Tompkins DM (2007) PCR detection of Plasmodium and blood meal identification in a native New Zealand mosquito. Journal of Vector Ecology 32: 154–156.B. MasseyDM GleesonD. SlaneyDM Tompkins2007PCR detection of Plasmodium and blood meal identification in a native New Zealand mosquito.Journal of Vector Ecology32154156
  8. 8. Gager AB, Loaiza JDR, Dearborn DC, Bermingham E (2008) Do mosquitoes filter the access of Plasmodium cytochrome b lineages to an avian host? Molecular Ecology 17: 2552–2561.AB GagerJDR LoaizaDC DearbornE. Bermingham2008Do mosquitoes filter the access of Plasmodium cytochrome b lineages to an avian host?Molecular Ecology1725522561
  9. 9. Ishtiaq F, Guillaumot L, Clegg SM, Phillimore AB, Black RA, et al. (2008) Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands. Molecular Ecology 17: 4545–4555.F. IshtiaqL. GuillaumotSM CleggAB PhillimoreRA Black2008Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands.Molecular Ecology1745454555
  10. 10. Ejiri H, Sato Y, Kim KS, Hara T, Tsuda Y, et al. (2011) Entomological Study on Transmission of Avian Malaria Parasites in a Zoological Garden in Japan: Bloodmeal Identification and Detection of Avian Malaria Parasite DNA From Blood-Fed Mosquitoes. Journal of Medical Entomology 48: 600–607.H. EjiriY. SatoKS KimT. HaraY. Tsuda2011Entomological Study on Transmission of Avian Malaria Parasites in a Zoological Garden in Japan: Bloodmeal Identification and Detection of Avian Malaria Parasite DNA From Blood-Fed Mosquitoes.Journal of Medical Entomology48600607
  11. 11. Ejiri H, Sato Y, Kim KS, Tsuda Y, Murata K, et al. (2011) Blood Meal Identification and Prevalence of Avian Malaria Parasite in Mosquitoes Collected at Kushiro Wetland, a Subarctic Zone of Japan. Journal of Medical Entomology 48: 904–908.H. EjiriY. SatoKS KimY. TsudaK. Murata2011Blood Meal Identification and Prevalence of Avian Malaria Parasite in Mosquitoes Collected at Kushiro Wetland, a Subarctic Zone of Japan.Journal of Medical Entomology48904908
  12. 12. Ejiri H, Sato Y, Sasaki E, Sumiyama D, Tsuda Y, et al. (2008) Detection of Avian Plasmodium spp. DNA Sequences from Mosquitoes Captured in Minami Daito Island of Japan. Journal of Veterinary Medical Science 70: 1205–1210.H. EjiriY. SatoE. SasakiD. SumiyamaY. Tsuda2008Detection of Avian Plasmodium spp. DNA Sequences from Mosquitoes Captured in Minami Daito Island of Japan.Journal of Veterinary Medical Science7012051210
  13. 13. Ejiri H, Sato Y, Sawai R, Sasaki E, Matsumoto R, et al. (2009) Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan. Parasitology Research 105: 629–633.H. EjiriY. SatoR. SawaiE. SasakiR. Matsumoto2009Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan.Parasitology Research105629633
  14. 14. Kim KS, Tsuda Y, Sasaki T, Kobayashi M, Hirota Y (2009) Mosquito blood-meal analysis for avian malaria study in wild bird communities: laboratory verification and application to Culex sasai (Diptera: Culicidae) collected in Tokyo, Japan. Parasitology Research 105: 1351–1357.KS KimY. TsudaT. SasakiM. KobayashiY. Hirota2009Mosquito blood-meal analysis for avian malaria study in wild bird communities: laboratory verification and application to Culex sasai (Diptera: Culicidae) collected in Tokyo, Japan.Parasitology Research10513511357
  15. 15. Carlson JS, Martinez-Gomez JE, Cornel A, Loiseau C, Sehgal RNM (2011) Implications of Plasmodium parasite infected mosquitoes on an insular avifauna: the case of Socorro Island, Mexico. Journal of Vector Ecology 36: 213–220.JS CarlsonJE Martinez-GomezA. CornelC. LoiseauRNM Sehgal2011Implications of Plasmodium parasite infected mosquitoes on an insular avifauna: the case of Socorro Island, Mexico.Journal of Vector Ecology36213220
  16. 16. Hughes T, Irwin P, Hofmeister E, Paskewitz SM (2010) Occurrence of Avian Plasmodium and West Nile Virus in Culex Species in Wisconsin. Journal of the American Mosquito Control Association 26: 24–31.T. HughesP. IrwinE. HofmeisterSM Paskewitz2010Occurrence of Avian Plasmodium and West Nile Virus in Culex Species in Wisconsin.Journal of the American Mosquito Control Association262431
  17. 17. Bensch S, Stjernman M, Hasselquist D, Ostman O, Hansson B, et al. (2000) Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proceedings of the Royal Society B-Biological Sciences 267: 1583–1589.S. BenschM. StjernmanD. HasselquistO. OstmanB. Hansson2000Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds.Proceedings of the Royal Society B-Biological Sciences26715831589
  18. 18. Bensch S, Waldenstrom J, Jonzen N, Westerdahl H, Hansson B, et al. (2007) Temporal dynamics and diversity of avian malaria parasites in a single host species. Journal of Animal Ecology 76: 112–122.S. BenschJ. WaldenstromN. JonzenH. WesterdahlB. Hansson2007Temporal dynamics and diversity of avian malaria parasites in a single host species.Journal of Animal Ecology76112122
  19. 19. Hellgren O, Krizanauskiene A, Valkiunas G, Bensch S (2007) Diversity and phylogeny of mitochondrial cytochrome B lineages from six morphospecies of avian Haemoproteus (Haemosporida : Haemoproteidae). Journal of Parasitology 93: 889–896.O. HellgrenA. KrizanauskieneG. ValkiunasS. Bensch2007Diversity and phylogeny of mitochondrial cytochrome B lineages from six morphospecies of avian Haemoproteus (Haemosporida : Haemoproteidae).Journal of Parasitology93889896
  20. 20. Christe P, Glaizot O, Strepparava N, Devevey G, Fumagalli L (in press) Twofold cost of reproduction: an increase in parental effort leads to higher malarial parasitaemia and to a decrease in resistance to oxidative stress. Proceedings of the Royal Society B-Biological Sciences. P. ChristeO. GlaizotN. StrepparavaG. DeveveyL. Fumagalliin pressTwofold cost of reproduction: an increase in parental effort leads to higher malarial parasitaemia and to a decrease in resistance to oxidative stress.Proceedings of the Royal Society B-Biological Sciences. doi: 10.1098/rspb.2011.1546. . doi: 10.1098/rspb.2011.1546.
  21. 21. Knowles SCL, Palinauskas V, Sheldon BC (2010) Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population. Journal of Evolutionary Biology 23: 557–569.SCL KnowlesV. PalinauskasBC Sheldon2010Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population.Journal of Evolutionary Biology23557569
  22. 22. Garnham PCC (1966) Malaria parasites and other Haemosporidia. Oxford: Blackwell Scientific Publications. PCC Garnham1966Malaria parasites and other HaemosporidiaOxfordBlackwell Scientific Publications
  23. 23. Fonseca DM, LaPointe DA, Fleischer RC (2000) Bottlenecks and multiple introductions: population genetics of the vector of avian malaria in Hawaii. Molecular Ecology 9: 1803–1814.DM FonsecaDA LaPointeRC Fleischer2000Bottlenecks and multiple introductions: population genetics of the vector of avian malaria in Hawaii.Molecular Ecology918031814
  24. 24. Belo NO, Pinheiro RT, Reis ES, Ricklefs RE, Braga EM (2011) Prevalence and Lineage Diversity of Avian Haemosporidians from Three Distinct Cerrado Habitats in Brazil. Plos One 6: NO BeloRT PinheiroES ReisRE RicklefsEM Braga2011Prevalence and Lineage Diversity of Avian Haemosporidians from Three Distinct Cerrado Habitats in Brazil.Plos One6
  25. 25. Vinogradova EB (2000) Mosquitoes Culex pipiens pipiens: taxonomy, distribution, ecology, physiology, genetics, applied importance and control. Sofia: Pensoft. EB Vinogradova2000Mosquitoes Culex pipiens pipiens: taxonomy, distribution, ecology, physiology, genetics, applied importance and controlSofiaPensoft
  26. 26. Waldenström J, Bensch S, Hasselquist D, Ostman O (2004) A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. Journal of Parasitology 90: 191–194.J. WaldenströmS. BenschD. HasselquistO. Ostman2004A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood.Journal of Parasitology90191194
  27. 27. Bensch S, Hellgren O, Perez-Tris J (2009) MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Molecular Ecology Resources 9: 1353–1358.S. BenschO. HellgrenJ. Perez-Tris2009MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages.Molecular Ecology Resources913531358
  28. 28. Zehtindjiev P, Križanauskienė A, Scebba S, Dimitrov D, Valkiūnas G, et al. (2012) Haemosporidian infections in skylarks (Alauda arvensis): a comparative PCR-based and microscopy study on the parasite diversity and prevalence in southern Italy and the Netherlands. European Journal of Wildlife Research 58: 335–344.P. ZehtindjievA. KrižanauskienėS. ScebbaD. DimitrovG. Valkiūnas2012Haemosporidian infections in skylarks (Alauda arvensis): a comparative PCR-based and microscopy study on the parasite diversity and prevalence in southern Italy and the Netherlands.European Journal of Wildlife Research58335344
  29. 29. Palinauskas V, Kosarev V, Shapoval A, Bensch S, Valkiunas G (2007) Comparison of mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites of the subgenera Haemamoeba and Giovannolaia (Haemosporida : Plasmodiidae). Zootaxa 39–50.V. PalinauskasV. KosarevA. ShapovalS. BenschG. Valkiunas2007Comparison of mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites of the subgenera Haemamoeba and Giovannolaia (Haemosporida : Plasmodiidae).Zootaxa3950
  30. 30. D'Amico VL, Baker AJ (2010) A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae). Journal of Ornithology 151: 951–954.VL D'AmicoAJ Baker2010A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae).Journal of Ornithology151951954
  31. 31. Martinsen ES, Perkins SL, Schall JJ (2008) A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): Evolution of life-history traits and host switches. Molecular Phylogenetics and Evolution 47: 261–273.ES MartinsenSL PerkinsJJ Schall2008A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): Evolution of life-history traits and host switches.Molecular Phylogenetics and Evolution47261273
  32. 32. Vezilier J, Nicot A, Gandon S, Rivero A (2010) Insecticide resistance and malaria transmission: infection rate and oocyst burden in Culex pipiens mosquitoes infected with Plasmodium relictum. Malaria Journal 9: J. VezilierA. NicotS. GandonA. Rivero2010Insecticide resistance and malaria transmission: infection rate and oocyst burden in Culex pipiens mosquitoes infected with Plasmodium relictum.Malaria Journal9
  33. 33. Lourenço-de-Oliveira R, de Castro FA (1991) Culex saltanensis Dyar, 1928 - natural vector of Plasmodium juxtanucleare in Rio de Janeiro, Brazil. Mem Inst Oswaldo Cruz, Rio de Janeiro 86: 87–94.R. Lourenço-de-OliveiraFA de Castro1991Culex saltanensis Dyar, 1928 - natural vector of Plasmodium juxtanucleare in Rio de Janeiro, Brazil.Mem Inst Oswaldo Cruz, Rio de Janeiro868794
  34. 34. Reiter ME, Lapointe DA (2007) Landscape factors influencing the spatial distribution and abundance of mosquito vector Culex quinquefasciatus (Diptera : Culicidae) in a mixed residential-agricultural community in Hawai'i. Journal of Medical Entomology 44: 861–868.ME ReiterDA Lapointe2007Landscape factors influencing the spatial distribution and abundance of mosquito vector Culex quinquefasciatus (Diptera : Culicidae) in a mixed residential-agricultural community in Hawai'i.Journal of Medical Entomology44861868
  35. 35. Njabo KY, Cornel AJ, Bonneaud C, Toffelmier E, Sehgal RNM, et al. (2011) Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest. Molecular Ecology 20: 1049–1061.KY NjaboAJ CornelC. BonneaudE. ToffelmierRNM Sehgal2011Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest.Molecular Ecology2010491061
  36. 36. Meyer CL, Bennett GF (1976) Observations on the sporogony of Plasmodium circumflexum Kikuth and Plasmodium polare Manwell in New Brunswick. Canadian Journal of Zoology 54: 133–142.CL MeyerGF Bennett1976Observations on the sporogony of Plasmodium circumflexum Kikuth and Plasmodium polare Manwell in New Brunswick.Canadian Journal of Zoology54133142
  37. 37. Thiemann TC, Reisen WK (2012) Evaluating Sampling Method Bias in Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae) Bloodmeal Identification Studies. Journal of Medical Entomology 49: 143–149.TC ThiemannWK Reisen2012Evaluating Sampling Method Bias in Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae) Bloodmeal Identification Studies.Journal of Medical Entomology49143149
  38. 38. Cerny O, Votypka J, Svobodova M (2011) Spatial feeding preferences of ornithophilic mosquitoes, blackflies and biting midges. Medical and Veterinary Entomology 25: 104–108.O. CernyJ. VotypkaM. Svobodova2011Spatial feeding preferences of ornithophilic mosquitoes, blackflies and biting midges.Medical and Veterinary Entomology25104108
  39. 39. Valkiunas G (2011) Haemosporidian vector research: marriage of molecular and microscopical approaches is essential. Molecular Ecology 20: 3084–3086.G. Valkiunas2011Haemosporidian vector research: marriage of molecular and microscopical approaches is essential.Molecular Ecology2030843086
  40. 40. Waldenström J, Bensch S, Kiboi S, Hasselquist D, Ottosson U (2002) Cross-species infection of blood parasites between resident and migratory songbirds in Africa. Molecular Ecology 11: 1545–1554.J. WaldenströmS. BenschS. KiboiD. HasselquistU. Ottosson2002Cross-species infection of blood parasites between resident and migratory songbirds in Africa.Molecular Ecology1115451554
  41. 41. Martinez-de la Puente J, Martinez J, Rivero-de Aguilar J, Herrero J, Merino S (2011) On the specificity of avian blood parasites: revealing specific and generalist relationships between haemosporidians and biting midges. Molecular Ecology 20: 3275–3287.J. Martinez-de la PuenteJ. MartinezJ. Rivero-de AguilarJ. HerreroS. Merino2011On the specificity of avian blood parasites: revealing specific and generalist relationships between haemosporidians and biting midges.Molecular Ecology2032753287
  42. 42. Megali A, Yannic G, Christe P (2011) Disease in the dark: molecular characterization of Polychromophilus murinus in temperate zone bats revealed a worldwide distribution of this malaria-like disease. Molecular Ecology 20: 1039–1048.A. MegaliG. YannicP. Christe2011Disease in the dark: molecular characterization of Polychromophilus murinus in temperate zone bats revealed a worldwide distribution of this malaria-like disease.Molecular Ecology2010391048
  43. 43. Tripet F (2009) Ecological Immunology of mosquito-malaria interactions: Of non-natural versus natural model systems and their inferences. Parasitology 136: 1935–1942.F. Tripet2009Ecological Immunology of mosquito-malaria interactions: Of non-natural versus natural model systems and their inferences.Parasitology13619351942