Life cycle studies and transmission mechanisms of myxozoan parasites Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander- Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Dennis Marc Kallert aus Erlangen Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 3. März 2006 Vorsitzender der Prüfungskommission: Prof. Dr. D.-P. Häder Erstberichterstatter: Prof. Dr. W. Haas Zweitberichterstatter: Prof. Dr. Dr. M. El-Matbouli -Contents- Contents ABBREVIATIONS 5 I. INTRODUCTION 6 1. The Phylum Myxozoa Grassé 1970: Common features 6 2. Current knowledge on myxozoan life cycles 8 3. Host invasion by actinospores 10 II. MATERIALS & METHODS 12 1. Animals and parasite cultivation 12 1.1. Fish for experimental infections 12 1.2. Oligochaetes 12 1.3. Parasites 13 1.4. Animals for substrate isolation 14 2. Experimental infections 14 2.1. Henneguya nuesslini Schuberg & Schröder 1905 14 2.2. Myxobolus parviformis sp.n. 16 3. Viability assay 17 4. Discharge experiments 18 4.1. Test substrates 18 4.2. Experimental set-up 19 4.2.1. Test for mechanical and chemical stimuli 19 4.2.2. Bulk experiments 20 4.2.3. Frequency dependency 21 4.2.4. Ca2+ dependency 22 5. Cinematography of polar filament discharge 22 6. Sporoplasm emission 22 7. Mucus fractionation 23 7.1. Heat and incubation 23 7.2. Ashing 23 7.3. Acetone precipitation 24 - 1 - -Contents- 7.4. Alcian blue precipitation 24 7.5. Fluorescamin derivatisation 24 7.6. Extraction with activated charcoal 25 7.7. Lipid extraction 25 7.7.1. Ether-extraction 25 7.7.2. Lipid isolation 26 7.8. Chemical fractionation 26 7.8.1. TFMS Deglycosylation 26 7.8.2. Sialic acid extraction 27 7.9. Enzymatic fractionation 28 7.10. Chromatographic fractionation 29 7.10.1. Ion-exchange 29 7.10.2. Lichroprep RP 18 30 8. Pure chemicals 30 9. Chemical analyses 32 9.1. Proteins 32 9.2. Amino compounds 32 9.3. Neutral sugars 32 9.4. Urea 32 9.5. Sialic acids 33 9.6. TLC-separation of lipid classes 33 9.7. Hydrophilic TLC 34 9.8. RP-HPLC 35 9.9. GC/MS 35 9.10. UV-Spectroscopy and fluorescence detection 36 9.11. HPIC-IPAD and HPLC-MDD 36 9.12. NMR-spectroscopy 37 10. Chemicals 38 11. Statistical methods 38 III. RESULTS 39 1. Life cycle experiments 39 1.1. Henneguya nuesslini 39 1.2. Myxobolus parviformis sp. n. 43 2. Viability assay 51 3. Polar filament discharge 51 - 2 - -Contents- 3.1. Visualisation of polar filament discharge 51 3.2. Mechanical and chemical stimuli 53 3.3. Experimental conditions for bulk experiments 53 3.4. Ca2+ dependency 54 3.5. Host specificity 55 3.5.1. Myxobolus cerebralis 55 3.5.2. Henneguya nuesslini 58 3.5.3. Myxobolus parviformis 58 3.6. Analysis of chemical signals for polar filament discharge 59 3.6.1. Ultrafiltration 59 3.6.2. Lipids 60 3.6.3. Amino compounds 61 3.6.4. Nucleotides 62 3.6.5. Carbohydrates 63 3.6.6. Proteins 68 3.6.7. Stability and small molecular compounds 70 3.6.8. Extraction with activated charcoal 72 3.6.9. Chromatographic fractionation 72 4. Sporoplasm emission 77 4.1. Emission time course 77 4.2. Dependence from discharge 78 4.3. Host specificity 78 4.4. Emission stimuli 78 5. Analyses 79 5.1. Substrate osmolality 79 5.2. Urea 80 5.3. Lipids 81 5.4. Hydrophilic TLC Analyses 83 5.5. Gradient chromatography 87 5.6. UV-Spectroscopy 89 5.7. HPIC-detection 89 5.8. Amino acids 91 5.9. Proteins 92 5.10. Carbohydrates 93 5.10.1. Neutral sugars 93 5.10.2. Sialic acids 94 5.10.3. GC-MS for monosaccharides 94 5.11. NMR-spectroscopy 95 - 3 - -Contents- IV. DISCUSSION 96 1. Myxozoan lifecycles 96 2. Host invasion by actinospores 103 3. Host signals for polar filament discharge 107 4. Impacts on myxozoan transmission 113 V. SUMMARY 116 VI. ZUSAMMENFASSUNG 118 VII. ACKNOWLEDGEMENTS 120 VIII. REFERENCES 121 IX. APPENDIX 130 - 4 - -Abbreviations- Abbreviations 1 J One bond AA Amino acid ATP Adenosine triphosphate BSA Bovine submaxillary mucin cAMP Cyclic adenosine monophosphate cGMP Cyclic guanosine monophosphate cIMP Cyclic inosine monophosphate D2O Deuterium oxide df Degrees of freedom FDA Fluorescein-diacetate GAGs Glycosaminoglycans GC/MS Gas chromatography/mass spectroscopy H & E Haematoxylin/eosin dye HMQC Hetero multiple quantum coherence HPIC-IPAD High performance ion exchange chromatography with integrated pulsed amperometric detection HPLC High performance liquid chromatography l Litres MDD Metal dye detection MW Molecular weight MWCO Molecular weight cut-off NBD-Cl 4-chloro-7-nitrobenzofurazan NMR Nuclear magnetic resonance p.e. Post exposure p.i. Post infection PBS Phosphate buffered saline PCR Polymerase chain reaction PI Propidium iodide RFLP Restriction fragment length polymorphism SD Standard deviation SDBS Data base system for organic compounds SEM Standard error of the mean SFW Standard fresh water TAM Triactinomyxon TFMS Trifluoromethanesulfonic acid TLC Thin-layer chromatography v/v Volume per volume w/v Weight per volume - 5 - -I. Introduction- I. Introduction 1. The Phylum Myxozoa Grassé 1970: Common features About 1350 species in 52 genera belong to the Myxozoa, an obligate parasitic group forming a separate phylum of multicellular metazoan parasites mainly of teleosts. Invertebrates like oligochaetes, bryozoans and polychaetes serve as secondary hosts. Despite being well-known as fish parasites, Myxozoa was also discovered in trematodes (Overstreet 1976, Siau et al. 1981), reptiles (Lom 1990) and amphibians (McAllister et al. 1995). Developmental stages were found in waterfowl (Lowenstine et al. 2002), in nervous systems of mammalians (Friedrich et al. 2000) and myxospores were even detected in human feces (Lebbad and Wilcox 1998, Moncada et al. 2001). The members of the most abundant genus, Myxobolus (Myxobolidae), have recently been reviewed by Eiras et al. (2005). Myxosporea were first described 1841 by Müller (named ‘Psorospermien’) and Štolc (1899) already noted the metazoan character of these organisms. Nevertheless, until the second half of the last century, the Myxozoa were commonly assigned to the protozoans. Due to the morphological variability within species and their highly reduced body organization, the taxonomy and the phylogenetic position of these obscure parasites are still subject of numerous controversies (Kent et al. 1994, 2001, Siddall et al. 1995, Anderson et al. 1998, Canning & Okamura 2004). An outgrowth of the ongoing phylogenetical work was the development of comprehensive PCR-based assays suitable for diagnostics. Although rather few species exert problematic symptoms, some members are severe pathogens of teleosts. Several species have a significant ecological and economic impact on freshwater and marine fish populations in Europe and the USA. Whirling disease, caused by the cosmopolitan parasite Myxobolus cerebralis, is still considered as one of the most devastating diseases among salmonid populations (Nehring & Walker 1996, Gilbert and Granath, 2003). It has been responsible for declines of the wild rainbow trout population in more than 22 northern american states (Hedrick et al. 1998). Other important pathogens of cultured fish include Tetracapsula bryosalmonae (proliferative kidney disease of salmonids) and Sphaerospora renicola (swimbladder inflammation of carp). Myxospores have been shown to resist freezing and passage through alimentary tracts of poikilothermic animals (e.g. birds, see El- - 6 - -I. Introduction- Matbouli and Hoffmann 1991) and are therefore highly adapted to environmental changes. Morphologically, the myxozoans share some unique features defining phylum affiliation. The class Malacosporea, probably an ancestral myxozoan group with few species described that partially resemble different (nemathelminth) characteristics, will be excluded in this study. Myxosporean specimens are uniformly small sized (myxospores 10-25 µm, actinospores up to 300µm), thereby showing an immense biodiversity in shape and morphological variation. Actinospores differ from myxospores by their triradial symmetry and the softer valves. Developmental stages lack cilia and cellular fission seems to occur without centrioles. Especially peculiar is the development including secondary and tertiary cell stages formed by endogeny during proliferation, a feature that is very rare in animals and otherwise found only in some protozoans. Most denominating are the polar capsules, specialised cell organelles reminiscent of cnidarian nemtocysts that are used for attachment to the host. Though recent findings revealed strong evidence for triploblast bilateral ancestors of the myxozoan phylum (Schlegel et al., 1996; Anderson et al. 1998; Okamura et al., 2002), there are arguments for a cnidarian origin of the polar capsules (discussed in Canning and Okamura, 2004). For example, the polar filaments were compared to the stinging threads of the parasitic cnidarian Polypodium hydriforme by Ibragimov (2001). Figure 1. Schematical structure of a myxozoan triactinomyxon-type actinospore. - 7 - -I. Introduction- 2. Current knowledge on myxozoan life cycles About 25 whole myxozoan life cycles have been elucidated today (see Kent et al. 2001). With few exceptions, they include two alternating hosts, an aquatic invertebrate (oligochaetes or bryozoans) and a vertebrate host, mainly teleost fish (Wolf & Markiw, 1984). Life cycle studies of myxozoans are difficult to conduct, most research has been carried out with M. cerebralis, which is available through continuous cultivation (El-Matbouli et al. 1992, Hedrick & El-Matbouli 2002).