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Khristianthesisresubmit.Pdf (7.238Mb) A Parasitic Dinoflagellate of the Ctenophore Mnemiopsis sp. by Khristian Deane Smith A thesis submitted to the Graduate Faculty of Auburn University in Partial fulfillment of the Requirements of the Degree of Masters of Science Auburn, Alabama December 12, 2011 Keywords: dinoflagellate, parasite, marine ctenophore, Mnemiopsis, Pentapharsodinium Copyright 2011 by Khristian Deane Smith Approved by Anthony Moss, Chair, Associate Professor of Biological Sciences Mark Liles, Assistant Professor of Biological Sciences Scott Santos, Associate Professor of Biological Sciences Abstract In this study I have sought to characterize a previously unknown parasitic dinoflagellate, which is associated with the costal ctenophore Mnemiopsis sp. Here, I describe its general morphology, based on an identification system created by Charles Kofoid used specifically for dinoflagellates. The identification system, Kofoid plate tabulation, allows for identification of genera or possibly species. The plate tabulation is used to interpret the gross morphological characters, number of thecal plates, and their arrangement. The study will also present on an overview of its parasitic relationship with the host and its reproductive capacity. Lastly, the study finishs with the phylogenetic placement based on rDNA, ITS, and cyt b molecular analysis. I conclude that the dinoflagellate’s phylogeny is placed tentatively into the genus Pentapharsodinium due to the inconsistencies within the monophyletic E/Pe clade. The life cycle of the dinoflagellate is characteristic of a parasite. However, the ability to successfully culture the dinoflagellate would suggest it is mixotrophic opportunistic parasite. ii Table of Contents Abstract............................................................................................................................... ii List of Tables ................................................................................................................vi-vii List of Figures...............................................................................................................viii-x Chapter 1: The Ctenophore Mnemiopsis leidyi and its Symbiotic Protist Assemblage, Ctenophore anatomy and placement ......................................................................1 Mnemiopis (host) ...................................................................................................2 Ctenophore parasites ..............................................................................................3 Dinoflagellate cortex ..............................................................................................5 Dinoflagellate corticotypes ....................................................................................6 Dinoflagellate morphological identification ...........................................................8 Nutritional diversity ..............................................................................................11 Dinoflagellate symbiosis ......................................................................................12 Parasitic dinoflagellates .........................................................................................14 Parasitic dinoflagellate reproduction ...................................................................18 A parasitic symbiont specifically associated with the ctenophore Mnemiopsis…19 Chapter 2: Collection and Culture of a Dinoflagellate Parasitic on the Ctenophore Mnemiopsis leidyi. .................................................................................................20 Introduction............................................................................................................20 Objectives and Rationale .......................................................................................21 iii Materials and Methods .........................................................................................21 Host Collection ......................................................................................................21 Dinoflagellate collection ......................................................................................23 Algal cultures .......................................................................................................24 Host reinfection ....................................................................................................25 Results ..................................................................................................................26 Algal cultures .......................................................................................................26 Reinfection experiment ........................................................................................27 Discussion and Conclusions ..............................................................................28 Chapter 3: Morphology of a Pentapharsodinium species parasitic on the ctenophore Mnemiopsis leidyi…………………………………………………………..…….31 Introduction............................................................................................................31 Objectives and Rationale .......................................................................................32 Materials and Methods .........................................................................................32 Microscopy ..........................................................................................................32 Fluorescence microscopy .....................................................................................33 Scanning Electron Microscopy and Transmission Electron Microscopy ............34 Results ..................................................................................................................35 Apical Plate Morphology.......................................................................................35 Cingulum Morphology .........................................................................................37 Antapical and Sulcal Morphology .......................................................................38 iv Parasitic Reproduction (the tomont) ......................................................................43 Discussion and Conclusions ..............................................................................44 Evidence for parasitism ........................................................................................46 Host specificity ....................................................................................................47 Chapter 4: Pentapharsodinium Molecular Analysis .........................................................48 Introduction............................................................................................................48 Objectives and Hypotheses ...................................................................................49 Materials and Methods .........................................................................................50 DNA extraction ....................................................................................................50 Polymerase Chain Reaction (PCR) and sequencing ............................................50 Sequence alignment and tree assembly ................................................................53 Results ..................................................................................................................60 18S rDNA analysis ..............................................................................................60 Internal transcribed spacer region analysis ..........................................................63 Molecular analysis cytochrome b (cty b) .............................................................65 Discussion and Conclusions ..................................................................................67 Overall conclusions ...........................................................................................................72 Acknowledgements ...........................................................................................................73 Appendix 1: Molecular Analysis of Trichodina ctenophorii and an unknown amoeba associated to the comb plates of Mnemiopsis .......................................................74 References .........................................................................................................................81 v List of Tables Table 1: Zoological Nomenclature and characteristics of parasitic dinoflagellate groups ............................................................................................................................................18 Table 2: Host collection sites and GPS coordinates .........................................................22 Table 3: List of 18S rDNA primers used in this study along with their specificity and references………………………………………………………………………………...52 Table 4: List of ITS primers used in this study along with their specificity and references………………………………………………………………………...53 Table 5: List of cty b primers used in this study along with their specificity and references……………………………………………………………………..….53 Table 6a: List of dinoflagellate species and GenBank accession number for SSU analysis…………………………………………………………………………...55 Table 6b: List of dinoflagellate species and GenBank accession number for SSU analysis…………………………………………………………………………...56 Table 7a: List of dinoflagellate species and GenBank accession number for ITS analysis…………………………………………………………………………...57
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