Parasitology Research

, Volume 100, Issue 6, pp 1311–1322 | Cite as

Linkage between mitochondrial cytochrome b lineages and morphospecies of two avian malaria parasites, with a description of Plasmodium (Novyella) ashfordi sp. nov

  • Gediminas Valkiūnas
  • Pavel Zehtindjiev
  • Olof Hellgren
  • Mihaela Ilieva
  • Tatjana A. Iezhova
  • Staffan Bensch
Original Paper


Numerous lineages of avian malaria parasites of the genus Plasmodium have been deposited in GenBank. However, only seven morphospecies have been linked to these lineages. This study linked two molecular sequences with morphospecies of malaria parasites. Two species of Plasmodium (mitochondrial cytochrome b gene lineages P-GRW2 and P-GRW4) were isolated from naturally infected adult great reed warblers (Acrocephalus arundinaceus) and inoculated to naive juvenile individuals of the same host species. Heavy parasitemia developed in the subinoculated birds, which enable identification of the species and deposition of their voucher specimens. Parasites of the lineage P-GRW2 were described as a new species, Plasmodium (Novyella) ashfordi, which is characterized primarily by the fan-like mature erythrocytic meronts containing seven to eight merozoites and the terminal position of clumped pigment granules in the gametocytes. Illustrations of the blood stages of the new species and Plasmodium (Haemamoeba) relictum (lineage P-GRW4) are given. The parasites of both lineages are transmitted in Africa and probably not in northern Europe. Other lineages closely related to P. ashfordi and P. relictum are identified. This study establishes the value of PCR-based identification of avian malaria parasites.



This article benefited from comments made by John R. Baker. The authors are grateful to Alan Warren, Natural History Museum, London for providing the type material of P. vaughani and P. relictum, and Robert Adlard, Queensland Museum, Queensland for providing the voucher blood slide of P. relictum capistranoae. The present study was supported in part by the Swedish Research Council, Carl Tryggers Foundation, and the Lithuanian State Science and Studies Foundation. The experiments described herein comply with the current laws of Sweden, Bulgaria, and Lithuania.


  1. Atkinson CT, van Riper C III (1991) Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon, and Haemoproteus. In: Loye JE, Zuk M (eds) Bird–parasite interactions: ecology, evolution, and behaviour, Oxford University Press, Oxford, pp 19–48Google Scholar
  2. Atkinson CT, Dusek RJ, Woods KL, Iko WM (2000) Pathogenicity of avian malaria in experimentally-infected Hawaii Amakihi. J Wildl Dis 36:197–204PubMedGoogle Scholar
  3. Beadell JS, Gering E, Austin J, Dumbacher JP, Peirce M, Pratt TK, Atkinson CT, Fleischer RC (2004) Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region. Mol Ecol 13:3829–3844PubMedCrossRefGoogle Scholar
  4. Beadell JS, Ishtiaq F, Covas R, Melo M, Warren BH, Atkinson CT, Bensch S, Graves GR, Jhala YV, Peirce MA, Rahmani AR, Fonseca DM, Fleischer RC (2006) Global phylogeographic limits of Hawaii’s avian malaria. Proc R Soc B 273:2935–2944PubMedCrossRefGoogle Scholar
  5. Bensch S, Åkesson S (2003) Temporal and spatial variation of hematozoans in Scandinavian willow warblers. J Parasitol 89:388–391PubMedCrossRefGoogle Scholar
  6. Bensch S, Stjernman M, Hasselquist D, Ostman O, Hansson B, Westerdahl H, Pinheiro RT (2000) Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proc R Soc Lond B 267:1583–1589CrossRefGoogle Scholar
  7. Bensch S, Pérez-Tris J, Waldensrtöm J, Hellgren O (2004) Linkage between nuclear and mitochondrial DNA sequences in avian malaria parasites: multiple cases of cryptic speciation? Evolution 58:1617–1621PubMedGoogle Scholar
  8. Bensch S, Waldenström J, Jonzén N, Westerdahl H, Hansson B, Sejberg D, Hasselquist D (2007) Temporal dynamics and diversity of avian malaria parasites in a single host species. J Anim Ecol 76:112–122PubMedCrossRefGoogle Scholar
  9. Bishop MA, Bennett GF (1992) Host–parasite catalogue of the avian haematozoa: supplement 1; Bibliography of the avian blood-inhabiting haematozoa: supplement 2. Mem Univ Newfoundland Occas Pap Biol 15:1–244Google Scholar
  10. Bonneaud C, Pérez-Tris J, Federici P, Chastel O, Sorci G (2006) Major histocompatibility alleles associated with local resistance to malaria in a passerine. Evolution 60:383–389PubMedGoogle Scholar
  11. Escalante AA, Freeland DE, Collins WE, Lal AA (1998) The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome. Proc Natl Acad Sci USA 95:8124–8129PubMedCrossRefGoogle Scholar
  12. Fallon S, Bermingham E, Ricklefs E (2003) Island and taxon effects in parasitism revisited: avian malaria in the Lesser Antilles. Evolution 57:606–615PubMedGoogle Scholar
  13. Feldman RA, Freed LA, Cann RL (1995) A PCR test for avian malaria in Hawaiian birds. Mol Ecol 4:663–673PubMedCrossRefGoogle Scholar
  14. Garnham PCC (1966) Malaria parasites and other Haemosporidia. Blackwell Scientific Publications, OxfordGoogle Scholar
  15. Garnham PCC, Duggan AJ (1986) Catalogue of the Garnham collection of malaria parasites and other Haemosporidia. The Wellcome Trust, LondonGoogle Scholar
  16. Godfrey RD, Fedynich AM, Pence DB (1987) Quantification of hematozoa in blood smears. J Wildl Dis 23:558–565PubMedGoogle Scholar
  17. Greiner EC, Bennett GF, White EM, Coombs RF (1975) Distribution of the avian hematozoa of North America. Can J Zool 53:1762–1787PubMedCrossRefGoogle Scholar
  18. Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites? Science 218:384–387PubMedCrossRefGoogle Scholar
  19. Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol 90:797–802PubMedCrossRefGoogle Scholar
  20. Iezhova TA, Valkiūnas G, Barlein F (2005) Vertebrate host specificity of two avian malaria parasites of subgenus Novyella: Plasmodium nucleophilum and Plasmodium vaughani. J Parasitol 91:472–474PubMedCrossRefGoogle Scholar
  21. Jarvi SI, Schultz JJ, Atkinson CT (2002) PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines. J Parasitol 88:153–158PubMedGoogle Scholar
  22. Kilpatrick AM, LaPointe DA, Atkinson CT, Woodworth BL, Lease JK, Reiter ME, Gross K (2006) Effects of chronic avian malaria (Plasmodium relictum) infection on reproductive success of Hawaii Amakihi (Hemignathus virens). Auk 123:764–774CrossRefGoogle Scholar
  23. Križanauskienė A, Hellgren O, Kosarev V, Sokolov L, Bensch S, Valkiūnas G (2006) Variation in host specificity between species of avian haemosporidian parasites: evidence from parasite morphology and cytochrome b gene sequences. J Parasitol 92:1319–1324PubMedCrossRefGoogle Scholar
  24. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  25. Laird M, van Riper C (1981) Questionable reports of Plasmodium from birds in Hawaii, with the recognition of P. relictum ssp. capistranoae. In: Canning EV (ed) Parasitological topics. A presentation volume to P. C. C. Garnham on the occasion of his 80th birthday, Special Publication No. 1, Allen, Lawrence, pp 159–165Google Scholar
  26. McClure HE, Poonswad P, Greiner EC, Laird M (1978) Haematozoa in the birds of Eastern and Southern Asia. Memorial University of Newfoundland, St. John’sGoogle Scholar
  27. McCutchan TF, Grim KC, Li J, Weiss W, Rathore D, Sullivan M, Graczyk TK, Kumar S, Cranfield MR (2004) Measuring the effects of an ever-changing environment on malaria control. Infect Immun 72:2248–2253PubMedCrossRefGoogle Scholar
  28. Nylander JAA (2004) mrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Software available at:
  29. Pérez-Tris J, Bensch S (2005) Diagnosing genetically diverse avian malaria infections using mixed-sequence analysis and TA-cloning. Parasitology 131:1–9CrossRefGoogle Scholar
  30. Perkins SL (2000) Species concepts and malaria parasites: detecting a cryptic species of Plasmodium. Proc R Soc Lond B 267:2345–2350CrossRefGoogle Scholar
  31. Perkins SL, Schall JJ (2002) A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences. J Parasitol 88:972–978PubMedGoogle Scholar
  32. Perkins SL, Osgood SM, Schall JJ (1998) Use of PCR for detection of subpatent infections in lizard malaria: implications for epizootiology. Mol Ecol 7:1587–1590CrossRefGoogle Scholar
  33. Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818PubMedCrossRefGoogle Scholar
  34. Rambaut A, Drummond A. TRACER: a program for analysing results from Bayesian MCMC programs such as BEAST & MrBayes. Available at
  35. Reullier J, Pérez-Tris J, Bensch S, Secondi J (2006) Diversity, distribution and exchange of blood parasites meeting at an avian moving contact zone. Mol Ecol 15:753–763PubMedCrossRefGoogle Scholar
  36. Ricklefs RE, Fallon SM (2002) Diversification and host switching in avian malaria parasites. Proc R Soc Lond B 269:885–892CrossRefGoogle Scholar
  37. Ricklefs RE, Fallon SM, Bermingham E (2004) Evolutionary relationships, cospeciation, and host switching in avian malaria parasites. Syst Biol 53:111–119PubMedCrossRefGoogle Scholar
  38. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  39. Russell PF (1932) Avian malaria studies, V. Plasmodium capistrani sp. nov., an avian malaria parasite in the Philippines. Philipp J Sci 48:269–289Google Scholar
  40. Schrenzel MD, Maalouf GA, Keener LL, Gaffney PM (2003) Molecular characterisation of malarial parasites in captive passerines birds. J Parasitol 89:1025–1033PubMedCrossRefGoogle Scholar
  41. Sehgal RNM, Jones HI, Smith TB (2005) Blood parasites of some West African birds. J Vet Med Sci 67:295–301PubMedCrossRefGoogle Scholar
  42. Unnasch TR, McLafferty M, Wirth DF (1985) The primary structure of the rRNA insertions of Plasmodium lophurae. Mol Biochem Parasitol 16:149–161PubMedCrossRefGoogle Scholar
  43. Valkiūnas G (2005) Avian malaria parasites and other haemosporidia. CRC, Boca RatonGoogle Scholar
  44. Valkiūnas G, Sehgal RNM, Iezhova TA, Smith TB (2005) Further observations on the blood parasites of birds in Uganda. J Wildl Dis 41:580–587PubMedGoogle Scholar
  45. Valkiūnas G, Bensch S, Iezhova TA, Križanauskienë A, Hellgren O, Bolshakov CV (2006) Nested cytochrome b PCR diagnostics underestimate mixed infections of avian blood hemosporidian parasites: microscopy is still essential. J Parasitol 92:418–422PubMedCrossRefGoogle Scholar
  46. van Riper III C, van Riper SG, Goff ML, Laird M (1986) The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecol Monogr 56:327–344CrossRefGoogle Scholar
  47. Waldenström J, Bensch S, Kiboi D, Hasselquist D, Ottosson U (2002) Cross-species infection of blood parasites between resident and migratory songbirds in Africa. Mol Ecol 11:1545–1554PubMedCrossRefGoogle Scholar
  48. Waldenström J, Bensch S, Hasselquist D, Östman Ö (2004) A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. J Parasitol 90:191–194PubMedCrossRefGoogle Scholar
  49. Westerdahl H, Waldenström J, Hansson B, Hasselquist D, von Schantz T, Bensch S (2005) Associations between malaria and MHC genes in a migratory songbird. Proc R Soc Lond B 272:1511–1518CrossRefGoogle Scholar
  50. Wiersch SC, Maier WA, Kampen H (2005) Plasmodium (Haemamoeba) cathemerium gene sequences for phylogenetic analysis of malaria parasites. Parasitol Res 96:90–94PubMedCrossRefGoogle Scholar
  51. Wilgenbusch JC, Warren DL, Swofford DL (2004) AWTY: a system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. Available at:

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Gediminas Valkiūnas
    • 1
  • Pavel Zehtindjiev
    • 2
  • Olof Hellgren
    • 3
  • Mihaela Ilieva
    • 2
  • Tatjana A. Iezhova
    • 1
  • Staffan Bensch
    • 3
  1. 1.Institute of EcologyVilnius UniversityVilnius 21Lithuania
  2. 2.Institute of ZoologyBulgarian Academy of SciencesSofiaBulgaria
  3. 3.Department of Animal Ecology, Ecology BuildingLund UniversityLundSweden

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