VILNIUS UNIVERSITY NATURE RESEARCH CENTRE Mikas ILGŪNAS Exo-erythrocytic development of avian malaria parasites and haemoproteids: completing the cycle DOCTORAL DISSERTATION Natural Sciences, Ecology and Environmental Sciences N 012 VILNIUS 2019 This dissertation was written between 2015 and 2019 at the Nature Research Centre. The research was supported by the Research Council of Lithuania. Academic supervisor: Habil. dr. Gediminas Valkiūnas (Nature Research Centre, Natural Sciences, Ecology and Environmental Sciences – N 012). This doctoral dissertation will be defended in a public meeting of the Dissertation Defence Panel: Chairman – Dr. Mindaugas Dagys (Nature Research Centre, Natural Sciences, Ecology and Environmental Sciences – N 012). Members: Prof. dr. Dennis Hasselquist (Lund University, Natural Sciences, Ecology and Environmental Sciences – N 012). Prof. habil. dr. Saulius Petkevičius (Lithuanian University of Health Sciences, Agriculture Sciences, Veterinary Medicine – A 002). Doc. dr. Elena Servienė (Nature Research Centre, Natural Sciences, Biology – N 010). Dr. Michael Tobler (Lund University, Natural Sciences, Ecology and Environmental Sciences – N 012). The dissertation shall be defended at a public meeting of the Dissertation Defence Panel at 10:00 on 11th December 2019 in meeting room 101 of the Nature Research Centre. Address: Akademijos str. 2 LT-08412, Vilnius, Lithuania Tel. +37052729257; e-mail: [email protected] The text of this dissertation can be accessed at the libraries of Nature Research Centre and Vilnius University, as well as on the website of Vilnius University: www.vu.lt/lt/naujienos/ivykiu-kalendorius VILNIAUS UNIVERSITETAS GAMTOS TYRIMŲ CENTRAS Mikas ILGŪNAS Egzoeritrocitinis paukščių maliarinių parazitų ir hemoproteidų vystymasis: gyvenimo ciklo papildymas DAKTARO DISERTACIJA Gamtos mokslai, Ekologija ir aplinkotyra N 012 VILNIUS 2019 Disertacija rengta 2015– 2019 metais Gamtos tyrimų centre. Mokslinis vadovas: Habil. dr. Gediminas Valkiūnas (Gamtos tyrimų centras, gamtos mokslai, ekologija ir aplinkotyra – N 012). Gynimo taryba: Pirmininkas – Dr. Mindaugas Dagys (Gamtos tyrimų centras, gamtos mokslai, ekologija ir aplinkotyra – N 012). Nariai: Prof. dr. Dennis Hasselquist (Lundo universitetas, gamtos mokslai, ekologija ir aplinkotyra – N012). Prof. habil. dr. Saulius Petkevičius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002) Doc. dr. Elena Servienė (Gamtos tyrimų centras, gamtos mokslai, biologija – N 010). Dr. Michael Tobler (Lundo Universitetas, gamtos mokslai, ekologija ir aplinkotyra – N 012). Disertacija ginama viešame Gynimo tarybos posėdyje 2019 m. gruodžio mėn. 11 d. 10 val. Gamtos tyrimų centras 101 posėdžių salėje. Adresas: Akademijos g. 2, LT-08412, Vilnius, Lietuva. Tel. +37052729257; e-mail: [email protected] Disertaciją galima peržiūrėti Gamtos tyrimų centro bei Vilniaus universiteto bibliotekose ir VU interneto svetainėje adresu: https://www.vu.lt/naujienos/ivykiu-kalendorius CONTENTS ABBREVIATIONS ............................................................................... 8 LIST OF PUBLICATIONS ON THE DISERTATION TOPIC ........... 9 AUTHOR CONTRIBUTIONS IN THE CORRESPONDING PAPERS .............................................................................................. 11 LIST OF CONFERENCE PRESENTATIONS ON THE SUBJECT OF THE DISSERTATION ................................................................. 13 AWARDS ............................................................................................ 14 SCIENTIFIC PROBLEM .................................................................... 15 OBJECTIVE AND MAIN TASKS OF THE STUDY ....................... 19 STATEMENTS TO BE DEFENDED ................................................ 20 NOVELTY OF THE STUDY ............................................................. 21 1. BRIEF LITERATURE OVERVIEW .............................................. 22 2. MATERIALS AND METHODS .................................................... 25 2.1. Study sites ................................................................................ 25 2.2. Sample collection ..................................................................... 25 2.2.1. Blood samples ................................................................... 25 2.2.2. Samples for histology research ......................................... 26 2.3. Chromogenic in situ hybridization .......................................... 26 2.4. Laser microdissection .............................................................. 27 2.5. Morphological analysis ............................................................ 27 2.6. Histological analysis ................................................................ 27 5 2.6.1. Haematoxylin-eosin stained preparations ......................... 27 2.6.2. Chromogenic in situ hybridization processed preparations ..................................................................................................... 28 2.7. Molecular analysis ................................................................... 28 2.7.1. DNA extraction from blood and polymerase chain reaction ..................................................................................................... 28 2.7.2. DNA extraction and PCR from single megalomeronts obtained by laser micro-dissection of histological preparations . 28 2.7.3. DNA sequencing ............................................................... 29 2.8. Phylogenetic analysis ............................................................... 29 2.9. Statistical analysis .................................................................... 30 2.10. Experimental design .............................................................. 30 2.11. Ethical statement .................................................................... 30 3. RESULTS AND DISSCUSSION ................................................... 32 3.1. Development of Plasmodium homocircumfexum (lineage pCOLL4) in common European passerine birds ............................ 32 3.2. Patterns of virulence of Plasmodium homocircumflexum (lineage pCOLL4) in experimentally exposed passerine birds .................... 33 3.3. Exo-erythrocytic development of Plasmodium elongatum (lineage pERIRUB01) .................................................................... 35 3.4. Exo-erythrocytic development of Plasmodium elongatum (lineage pGRW6) ............................................................................ 35 3.5. Exo-erythrocytic development of Haemoproteus majoris (lineages hPHYBOR04 and hPARUS1) ......................................... 37 6 3.6. Exo-erythrocytic development of Plasmodium relictum (lineage hPHCOL01) .................................................................................... 39 3.7. Exo-erythrocytic development of Plasmodium delichoni (lineage pCOLL6) ......................................................................................... 40 3.8. Exo-erythrocytic development of Haemoproteus ciconiae ..... 40 CONCLUSIONS ................................................................................. 42 SCIENTIFIC AND PRACTICAL SIGNIFICANCE .......................... 43 SANTRAUKA .................................................................................... 44 REFERENCES .................................................................................... 63 ADDITIONAL PAPERS PUBLISHED DURING THE PHD STUDIES (NOT INCLUDED IN THIS THESIS).............................. 74 FUNDING ........................................................................................... 76 ACKNOWLEDGEMENTS ................................................................ 77 CURRICULUM VITAE ..................................................................... 80 COPIES OF PUBLICATIONS ........................................................... 85 7 ABBREVIATIONS BCIP – 5-bromo-4-chloro-3-indolyl phosphate DNA – deoxynucleic acid dpe – days post exposure H&E – haematoxylin and eosin ISH – chromogenic in situ hybridization NBT – 4-nitro blue tetrazolium chloride PCR – polymerase chain reaction rRNA – ribosomal ribonucleic acid 8 LIST OF PUBLICATIONS ON THE DISERTATION TOPIC The dissertation is based on the following co-authored original publications in journals with an impact factor and referred in the Clarivate Analytics Web of Science database. Throughout the text, the publications are referred using the Roman numerals: I. Ilgūnas, M., Bukauskaitė, D., Palinauskas, V., Iezhova, T.A., Dinhopl, N., Nedorost, N., Weissenbacher-Lang, C., Weissenböck, H., Valkiūnas, G. 2016. Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites. Malaria Journal 15, 256. doi: 10.1186/s12936-016-1310-x. (Parasitology Q2; Tropical Medicine Q1) II. Palinauskas, V., Žiegytė, R., Iezhova, T.A., Ilgūnas, M., Bernotienė, R., Valkiūnas, G. 2016. Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite. International Journal for Parasitology 46, 697-707. doi: 10.1016/j.ijpara.2016.05.005. (Parasitology Q1) III. Valkiūnas, G., Ilgūnas, M., Bukauskaitė, D., Iezhova, T.A. 2016. Description of Haemoproteus ciconiae sp. nov. (Haemoproteidae, Haemosporida) from the white stork Ciconia ciconia, with remarks on insensitivity of established polymerase chain reaction assays to detect this infection. Parasitology Research