Laser capture microdissection microscopy and genome sequencing of the avian malaria parasite, Plasmodium relictum
- 423 Downloads
Acquiring genomic material from avian malaria parasites for genome sequencing has proven problematic due to the nucleation of avian erythrocytes, which produces a large ratio of host to parasite DNA (∼1 million to 1 bp). We tested the ability of laser capture microdissection microscopy to isolate parasite cells from individual avian erythrocytes for four avian Plasmodium species, and subsequently applied whole genome amplification and Illumina sequencing methods to Plasmodium relictum (lineage pSGS1) to produce sequence reads of the P. relictum genome. We assembled ∼335 kbp of parasite DNA from this species, but were unable to completely avoid contamination by host DNA and other sources. However, it is clear that laser capture microdissection holds promise for the isolation of genomic material from haemosporidian parasites in intracellular life stages. In particular, laser capture microdissection may prove useful for isolating individual parasite species from co-infected hosts. Although not explicitly tested in this study, laser capture microdissection may also have important applications for isolation of rare parasite lineages and museum specimens for which no fresh material exists.
KeywordsAvian malaria Plasmodium genome laser capture microdissection microscopy
This project was completed with support from the Athena Fund, the Cornell Lab of Ornithology, and the Cornell College of Veterinary Medicine (Ithaca, NY, USA), as well as by the Negaunee Foundation at the Field Museum of Natural History (Chicago, IL, USA). Funding was also provided by the European Social Fund under the global grant measure (VPI-3.1.-ŠMM-07-K-01-047) to the Lithuania Research Council. We would like to thank Johanna Dela Cruz, Paulina Pavinski Bitar, and Peter Schweizer for their assistance with technical aspects of LCMM, DNA extraction, and genome sequencing, respectively. We also thank the Wellcome Trust Sanger Institute for access to data from an unpublished Plasmodium relictum genome (raw data available via NCBI Accession Number PRJEB2579).
Compliance with ethical standards
Experimental procedures for this study were approved by the Ethical Commission of the Baltic Laboratory Animal Science Association (Lithuania) and Lithuanian State Food and Veterinary Office (Ref. no. 2012/01/04-0221), Lithuania. Experimental procedures conducted in the USA complied with IACUC permit 17601 UC Davis.
- Bancroft JD (1967) An introduction to histochemical technique. Butterworth & Co., LondonGoogle Scholar
- Clark NJ, Clegg SM, Lima MR (2014) A review of global diversity in avian haemosporidians (Plasmodium and Haemoproteus: Haemosporida): new insights from molecular data. International Journal for Parasitology. doi: http://dx.doi.org/10.1016/j.ijpara.2014.01.004
- Garnham PCC (1966) Malaria parasites and other Haemosporidia. Blackwell, OxfordGoogle Scholar
- Ishtiaq F, Beadell JS, Warren BH, Fleischer RC (2011) Diversity and distribution of avian haematozoan parasites in the western Indian Ocean region: a molecular survey. J Parasitol FirstView:1-11 doi: 10.1017/S0031182011001831
- Lutz HL, Hochachka WM, Engel JI, Bell JA, Tkach VV, Bates SJ, Weckstein JD (2015) Parasite prevalence corresponds to host life history in a diverse assemblage of afrotropical birds and haemosporidian parasites. PLoS One 10:e0121254. doi: 10.1371/journal.pone.0121254 CrossRefPubMedPubMedCentralGoogle Scholar
- Lutz HL, Patterson BD, Kerbis Peterhans JC, Stanley WT, Webala PW, Gnoske TP, Hackett SJ, Stanhope MJ (2016) Diverse sampling of East African haemosporidians reveals chiropteran origin of malaria parasites in primates and rodents. Mol Phylogenet Evol 99:7–15. doi: 10.1016/j.ympev.2016.03.004 CrossRefPubMedGoogle Scholar
- Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, Prjibelski AD, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, Clingenpeel SR, Woyke T, Mclean JS, Tesler G, Alekseyev MA (2013) Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol 20:714–737. doi: 10.1089/cmb.2013.0084 CrossRefPubMedPubMedCentralGoogle Scholar
- Palinauskas V, Žiegytė R, Ilgūnas M, Iezhova TA, Bernotienė R, Bolshakov C, Valkiūnas G (2015) Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes. Int J Parasitol 45:51-62 doi: http://dx.doi.org/10.1016/j.ijpara.2014.08.012
- Prugnolle F, Durand P, Neel C, Ollomo B, Ayala FJ, Arnathau C, Etienne L, Mpoudi-Ngole E, Nkoghe D, Leroy E, Delaporte E, Peeters M, Renaud F (2010) African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum. Proc Natl Acad Sci U S A 107:1458–1463CrossRefPubMedPubMedCentralGoogle Scholar
- Stevens A, Francis RJ (1996) Micro-organisms. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques. Churchill Livingstone, New YorkGoogle Scholar
- Telford SR (2009) Haemoparasites of the Reptilia: color atlas and text. CRC Press, Boca RatonGoogle Scholar
- Valkiūnas G (2005) Avian malarial parasites and other Haemosporidia. CRC Press, Boca RatonGoogle Scholar