VILNIUS UNIVERSITY NATURE RESERCH CENTRE RITA ŽIEGYTĖ THE EXPERIMENTAL STUDY ON DEVELOPMENT OF AVIAN MALARIA PARASITES (PLASMODIUM) AND HAEMOPROTEIDS (HAEMOPROTEUS) IN VECTORS Doctoral Dissertation Biomedical Sciences, Zoology (05 B) Vilnius, 2014 The research was carried out at Nature Research Centre during 2010 – 2014. Research Supervisor: dr. habil. Gediminas Valkiūnas (Nature Research Centre, Biomedical sciences, Zoology – 05 B) Consultant Supervisor: prof. dr. Staffan Bensch (Department of Animal Ecology of Lund University, Biomedical sciences, Zoology – 05 B) CONTENTS 1. LITERATURE REVIEW ......................................................................................12 1.1. Brief description of the life cycle of Haemoproteus and Plasmodium (Haemosporida) parasites in blood - sucking dipteran insects ..........................12 1.2. Review of studies of Haemoproteus and Plasmodium parasites in blood - sucking insects related to the dissertation ................................................15 1.2.1. Brief historical review on determining vectors of avian Haemoproteus and Plasmodium parasites ......................................................15 1.2.2. Investigation of sporogony of the Plasmodium relictum genetic lineages in mosquitoes Culex pipiens pipiens ....................................17 1.2.3. Investigation of sporogony of the Plasmodium relictum genetic lineages in mosquitoes Culex pipiens pipiens form molestus ..........18 1.2.4. Haemoproteus spp. sporogony in the biting midges ...........................18 1.2.5. Development of Haemoproteus parasites in mosquitoes ....................19 1.2.6. The virulence of Haemoproteus infections to blood - sucking insects ....................................................................................................21 1.2.7. Sporogonic development of Plasmodium homocircumflexum (lineage COLL4) in mosquitoes ....................................22 2. MATERIALS AND METHODS .........................................................................23 2.1. Study site .............................................................................................................23 2.2. Experiment 1: Determination of sporogony of Plasmodium relictum (lineages pSGS1 and pGRW11) in mosquitoes Culex pipiens pipiens ...............24 2.2.1. The lineages of Plasmodium relictum ....................................................24 2.2.2. Experimental canaries and maintenance of Plasmodium relictum in birds ..................................................................................................24 2.2.3. Collection of blood samples and their microscopic and PCR - based examination ..................................................................................25 2.2.4. Phylogenetic analysis ...............................................................................26 2.2.5. Collection, maintainance and identification of mosquitoes ..............26 2.2.6 Experimental infection of mosquitoes ...................................................27 2.2.7. Dissection of mosquitoes and making preparations of ookinetes, oocysts and sporozoites ..............................................................28 2.2.8. Microscopic examination of vector preparations and parasite morphology ..........................................................................................29 3 2.2.9. Infection of canaries by bites of experimentally infected mosquitoes ...........................................................................................................29 2.3. Experiment 2: Determination of sporogony of Plasmodium relictum (lineages pSGS1 and pGRW11) in mosquitoes Culex pipiens pipiens form molestus ...................................................................................................................29 2.3.1. The lineages of Plasmodium relictum ....................................................29 2.3.2. Experimental canaries and maintenance of Plasmodium relictum in birds ..................................................................................................30 2.3.3. Collection of blood samples and their microscopic and PGR - based examination has been described in paragraph 2.2.3. ..........................30 2.3.4. Establishment of mosquitos colony and experimental infection of mosquitoes ......................................................................................................30 2.3.5. PCR - based examination ........................................................................30 2.3.6. Statistical analysis .....................................................................................31 2.3.7. Dissection of mosquitoes and making preparations of ookinetes, oocysts and sporozoites ..............................................................31 2.4. Experiment 3: Determination of the vectors of Haemoproteus minutus and Haemoproteus belopolskyi (Haemoproteidae) ...............................................31 2.4.1. Study site, collection of blood samples and experimental birds ........31 2.4.2. Wild - caught biting midges ...................................................................32 2.4.3. Experimental design ................................................................................32 2.4.4. Dissection of biting midges and making preparations of ookinetes, oocysts and sporozoites ..............................................................33 2.4.5. Microscopic examinations of preparations parasite morphology and statistical analysis .................................................................33 2.4.6. PCR - based examination ........................................................................34 2.5. Experiment 4: Determination peculiarities of the development of Haemoproteus parasites in blood - sucking mosquitoes (Culicidae) .................34 2.5.1. Study site and collection of bird blood samples ...................................34 2.5.2.Experimental design .................................................................................35 2.5.3. PCR - based examination and sequencing ...........................................36 2.5.4. Dissection of mosquitoes and making preparations of ookinetes, oocysts and sporozoites ..............................................................37 2.5.5. Examination of preparations, voucher material and statistical analysis ................................................................................................37 4 2.6. Experiment 5: Determination of virulence of Haemoproteus parasites for bird - biting mosquitoes and patterns of their development in these insects ................................................................................................................37 2.6.1. Collection of blood samples ....................................................................37 2.6.2. Experimental design ................................................................................37 2.6.3. PCR - based examination and sequencing ...........................................39 2.6.4. Dissection of mosquitoes and making preparations of parasites ......39 2.6.5. Examination of preparations, voucher material and statistical analysis .................................................................................................................40 2.7. Experiment 6: Determination of development of Plasmodium homocircumflexum (lineage COLL4) in mosquitoes Culex pipiens pipiens, Culex pipiens pipiens form molestus and Aedes vexans. ......................................40 2.7.1. Plasmodium homocircumflexum lineage and experimental infections of birds ...............................................................................................40 2.7.2. Collection of blood from exposed canaries ..........................................41 2.7.3. Collection, maintenance and experimental infection of mosquitoes .....41 2.7.4. PCR - based examination ........................................................................41 2.7.5. Microscopy, morphological analysis of parasites and statistical analysis ................................................................................................42 2.7.6. Dissection of mosquitoes and preparations of ookinetes, oocysts and sporozoites specimens ..................................................................42 3. RESULTS AND DISCUSSION ...........................................................................43 3.1. Experiement 1. Patterns of development of the closely related lineages pSGS1 and pGRW11 of Plasmodium relictum and different isolate of the lineage pSGS1 in the blood sucking mosquitoes Culex pipiens pipiens (Culicidae) ..................................................................................................................43 3.1.1. Results ........................................................................................................43 3.1.2. Discussion .................................................................................................45 3.2. Experiment 2. The developmend of Plasmodium relictum (pSGS1 and pGRW11) in mosquito Culex pipiens pipiens form molestus .....................48 3.2.1. Results ........................................................................................................48 3.2.2. Discussion .................................................................................................49 3.3. Experiment 3. Determination of the vectors of Haemoproteus minutus and H. belopolskyi ...................................................................................................51