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 Table 7b: List of dinoflagellate species and GenBank accession number for ITS analysis????????????????????????????...58 Table 8: List of dinoflagellate species and GenBank accession number for Cty b analysis???????????????????????????...?59 Table 9: Consensus sequence for 18S rDNA gene sequences from the parasitic dinoflagellate isolate???????????????????????..61 Table 10: Consensus sequence for ITS sequences from the parasitic dinoflagellate isolate?????????????????????????????.63 Table 11: Consensus sequence for cyt b gene sequences from the parasitic dinoflagellate isolate????????????????????????????.?65 vi Table 12: Contiguous sequence of Trichodina ctenophorii using primer 18SCOMF1?.75 Table 13: Contiguous sequences of Trichodina ctenophorii using primer 18SCOMR1...75 Table 14: List of Trichodina species and GenBank accession numbers???????75 Table 15: List of PCR primes for Flabellula ? like gymnamoebae analysis?????.77 Table 16: Contiguous sequences of Flabellula ? like gymnamoebae using primer 18SCOMF1?????????????????????????...?.78 Table 17: Contiguous sequences of Flabellula ? like gymnamoebae using primer 23FPL?????????????????????????????.79 Table 18: Contiguous sequences of Flabellula ? like gymnamoebae using primer 518R?????????????????????????????...80 vii List of Figures Figures 1: Ctenophore (Mnemiopsis) Gross Anatomy ........................................................1 Figures 2: Ectoparasites of Mnemiopsis .............................................................................4 Figures 3: Dinoflgellate Amphiesma...................................................................................5 Figures 4: Armored vs. Unarmored Dinoflagellate ............................................................6 Figures 5: Dinoflagellate Corticotypes ................................................................................7 Figures 6: Dinoflagellate Plate Designation .......................................................................8 Figures 7: Dinoflagellate Flagellar Arrangement ...............................................................9 Figures 8: Types of dinoflagellate cingular displacementIn .............................................10 Figures 9: Ectoparasitic Dinoflagellate Attachment..........................................................12 Figures 10: Diagram of the Life Cycle of Amoebophrya ..................................................17 Figures 11: Light Micrograph of Mnemiopsis with high dinoflagellate surface density ..23 Figures 12: Growth Curve of Cultured Dinoflagellates.....................................................26 Figures 13: Reinfection Experiment Mortality Chart .......................................................27 Figures 14: SEM of Dinoflagellate Surface Texture .........................................................35 Figures 15: 1000x Light Micrograph of Parasitic Dinoflagellate Showing Ortho conformation .........................................................................................................36 Figures 16: Calcofluor White Staining Under UV Fluorescence Showing hexa conformation..........................................................................................................36 viii Figures 17: Calcofluor White Staining Under UV Fluorescence Showing Homologous Cingular Sutures.....................................................................................................37 Figures 18: Calcofluor White Staining Under UV Fluorescence Showing Antapical Plate Morphology ...........................................................................................................38 Figures 19: Diagrama of Known Pentapharsodinium Species Depicting Antapical Plate Morphology ...........................................................................................................38 Figures 20: Calcofluor White Staining Under UV Fluorescence Showing Sulcal Region ????????????????????????????????40 Figures 21: Dinoflagellate Cells Associated with Host .....................................................41 Figures 22: Dinoflagellate Peduncle..................................................................................41 Figures 23 Light micrograph Series Showing Parasitic Reproduction, Termed Palintomy After Disassociation with Host ..............................................................................42 Figures 24: In situ SEM of Host and Dinoflagellates .......................................................44 Figures 25: Diagram Depicting the Sulcal Region of an Ensiculifera Species .................45 Figures 26: SSU rDNA Maximum Likelihood Search with 1,000 Replicates Using GTR Model .....................................................................................................................62 Figures 27: ITS Maximum Likelihood Search with 1,000 Replicates Using GTR Model???? .....................................................................................................64 Figures 28: Cyt b Maximum Likelihood Search with 1,000 Replicates Using GTR Model?????????????????????????????.66 ix x Figures 29: Maximum Likelihood Phylogeny for Calcareous Dinoflagellates (Gottschling) .........................................................................................................70 Figures 30: ITS Analysis Using Neighbor Model (D?Onofrio).........................................71 Figures 31: Chronogram Depiction the Separation of the E/Pe Clade (D?Onofrio)..........71 Figures 32: Neighbor Joining Tree of Trichodina showing that Trichodina Ctenophorii sits within the Trichodina group............................................................................76 Chapter 1: Introduction. The Ctenophore Mnemiopsis leidyi and its Symbiotic Protist Assemblage. Ctenophore anatomy and placement Comb jellies, or ctenophores (Phylum Ctenophora) are gelatinous marine planktonic organisms found throughout the oceans of the world. All are predators distinguished from Phylum Cnidaria by the lack of nematocysts, an oral-aboral body axis that positions the aboral organ and mouth at the most distant positions on the body, and eight rows of ciliary paddles called ctenes or comb plates, from which the group derives its name. The paddles are used for locomotion and food acquisition via fluid transport driven by ciliary beating (Colin, Costello et al. 2010). Ctenophores contain an extensive mesoglea that consists mostly of acellular components and water for structural support Figure 1. Ctenophore (Mnemiopsis) Gross Anatomy Subsagittal plates/rows Lobes Subtentacular plates/rows Auricles Tentacular bulb Statocyst Food groove 1 (Harbison 1985). Ctenophores undergo embryological development via a distinctive, determinate series of cell divisions (Martindale and Henry 1999). All possess a single aborally positioned statocyst. Members of Class Tentaculata possess tentacles bearing specialized adhesive cells at some stage in their life cycle; all members of Class Nuda never bear tentacles at any stage. Ctenophores are usually hermaphroditic, storing both gametes beneath their comb rows (Pang and Martindale 2008). Recent molecular phylogenies of ctenophores (Podar, Haddock et al. 2001; Dunn, Hejnol et al. 2008; Hejnol, Obst et al. 2009) agree with previous classical analyses (Hyman 1940) and place them into Phylum Ctenophora, which has approximately greater than 150 known species (Mills 2007) broken into two classes, Tentaculata and Nuda. Mnemiopsis The coastal ctenophore Mnemiopsis leidyi (Class Tentaculata; Order Lobata; (Fig. 1) is endemic to the Western Atlantic. It is a planktonic predator found from the Bay of Campeche along the North American Atlantic coast as far north as southern New England (Purcell, Shiganova et al. 2001). Mnemiopsis routinely experiences transatlantic transport to the Old World via ship?s ballast water (Harbison and Volovik 1993; Ruiz, Carlton et al. 1997; Ivanov, Kamakin et al. 2000; Bai, Zhang et al. 2005). Mnemiopsis is an exceptionally invasive organism, and in the 1980s invaded the Black Sea; in the 1990s, the Caspian; in 2006, the North and Baltic Seas, and most recently the Western Mediterranean. Like many invasive organisms, Mnemiopsis has severely impacted the ecology of numerous bays and estuaries in invaded regions. The lack of the naturally occurring New World predators 2 Bero? ovata and Chrysaora quinquecirrha (Burrell and Van Engel 1976; Finenko, Anninsky et al. 2001) inevitably results in swarms of overwhelming magnitude. Invasive Mnemiopsis populations are typically regulated by local seasonality and their inability to survive sustained water temperatures below 2 ?C. Invasions of Mnemiopsis into the Black Sea and Caspian Sea caused multibillion-dollar losses to local fisheries and exacted tremendous ecological damage (Ivanov, Kamakin et al. 2000; Purcell, Shiganova et al. 2001; Kideys, Roohi et al. 2005). Damage resulting from invasions into the North and Baltic Seas has yet to be assessed (Hansson 2006). Microsatellite analyses have recently revealed that Mnemiopsis invaded the Old World in two waves (Reusch, Bolte et al.) Ctenophore parasites Ctenophores are known to often carry a multitude of parasites, some which may use ctenophores as an intermediate host (Purcell and Arai 2001). Several species of parasitic nematodes (K?ie 1993; Gayevskaya and Mordvinova 1994) have been observed to be associated with ctenophores. Trematodes (Yip 1984) are well established as a known ctenophore parasite and even a cnidarian (Bumann and Puls 1996). In the mid 1990s the Moss laboratory reported that Mnemiopsis leidyi of Mobile Bay and the Northern Gulf of Mexico harbored an assemblage of protistan symbionts (Moss, Estes et al. 2001). Four distinct organisms were observed: a trichodine (Fig. 2a) (Estes, Reynolds et al. 1997), two types of amoebae (Fig. 2b) (Smith, Versteeg, Rogerson, Gast and Moss, in preparation), and a large ectodermally attached dinoflagellate (Fig. 2c). The trichodine, Trichodina ctenophorii, preferentially attaches to the host at the aboral side of the auricular and locomotary comb plates. T. ctenophorii appears to have a commensal relationship with Mnemiopsis. A Flabellula ? like gymnamoebae only appears on the 3 comb plates and forms a parasitic relationship with Mnemiopsis. A Vexillifera ?like commensal gymnamoebae is found at low densities on the ctenophore ectoderm (Moss, Estes et al. 2001). The dinoflagellate can be found attached to the ectoderm, embedded in the mesoglea, and freely swimming in the vascular canals. Here, I provide evidence that the dinoflagellate is a parasite. Moss and colleagues suggested that the dinoflagellate was indeed parasitic because it caused localized collapse of the mesoglea, particularly in regions near the aboral pole and where there were relatively large numbers of cells. c b a Figure 2. a) Trichodina ctenophorii attached to Mnemiopsis comb plate. Scale: 10?m b) Flabellulid gymnamoebae attached to Mnemiopsis comb plate. Scale: 5?m. c) Dinoflagellate attached to Mnemiopsis. Open arrow indicates longitudinal flagellum, asterisk indicates cingulum, and white arrow indicates transverse flagellum. Scale: 20?m (Moss, Estes et al. 2001) 4 Dinoflagellate cortex The dinoflagellate cortex, also called the theca or amphiesma, is comprised of a plasmalemma, a thin peripheral cytoplasmic layer, a single layer of flattened cortical (amphiesmal) vesicles, and a layer of microtubules located within the cytoplasm beneath the cortical vesicles (Dodge and Crawford 1970). Another layer may also be present: termed the pellicular layer or pellicle, this fourth layer may be found under the Figure 3. Dinoflagellate amphiesma depicting cortical layers (Kwok and Wong 2003). The outermost membrane is the plasma membrane. dinoflagellate cell cortex. TEM studies performed by Dodge and Crawford (Dodge and Crawford 1970) the cortical vesicles may be filled with liquid, flocculent or granular material, a continuous sheet of dense material, or a thick rigid plate. Dense cellulosic plates observed within the cortical vesicles are the basis for armored dinoflagellates morphological plate tabulations (Fig. 3). 5 The structure, life styles, and life stages of dinoflagellates are very diverse. Dinoflagellate cell size can range from as small as 10?m to as large as 2.0mm. Dinoflagellate morphological taxonomy is based on the presence or absence of cellulosic plates, plate thickness, and plate outline. Morphology-based taxonomy is also based on very thin, transient, precursor thecal membranes thought to be precursors to formation of the thecal plates. Dinoflagellate corticotypes Dinoflagellates can be divided morphologically into five corticotypes based on the structure of the theca (Taylor 1980). The most common corticotypes are the gymnodinoids, peridinoids, gonyaulacoids, dinophysoids, and prorocentroids; however, a sixth corticotype, woloszynskoids, has been proposed (Netzel and D?rr 1984). 10?m a b Figure 4. a)Pentapharsodinium tyrrhenicum is an example of an armored dinoflagellate containing cellulose within its thecal vesicles (Gottschling, Keupp et al. 2005). b) Karenia brevis is an example of an unarmored or naked dinoflagellate (Haywood, Steidinger et al. 2004). The prorocentroid corticotype (Fig. 5) is exemplified by dinoflagellates of the genus Prorocentrum. The prorocentroid amphiesma is made from two large plates, referred to as valves that usually possess trichocyst pores. Also a number of thecal plates 6 surround the flagellar pores (Fig. 6), periflagellar plates. The number of periflagellar plates range from eight to twelve, and is species-dependent. The total plate count in dinophysoids is generally eighteen to nineteen plates (Balech 1980). The dinophysoid theca (Fig. 5) is very similar to that of prorocentroids, with regards to having two valves and numerous periflagellar plates. In addition to this dinophysoids have a four plate cingular girdle (Fig. 6) and a sulcal region (Fig. 6). Gymnodinoids and woloszynskoids are generally referred to as the unarmored dinoflagellates (Fig. 4b). However, various gymodinoid and woloszynskoid species are known to possess very thin plate structures within their cortical vesicles (Dodge and Crawford 1969; Schnepf and Deichgr?ber 1972). The gonyaulacoid and peridinioid corticotypes belong to the armored morphotypes (Fig. 4a and 5). The armored dinoflagellates possess five latitudinal plate series, apicals, precingulars, cingulars, postcingulars, and antapicals. Another non- latitudinal series, the sulcals, and an apical pore complex, or APC, is also present (Taylor 1987). Any additional plates are referred to as intercalary plates. Valve Figure 5. Dinoflagellate corticotypes (Lee, Hutner et al. 1985) FP, VP, and AP indicates the flagellar pore, ventral pore, and the accessory pore respectively. 7 Dinoflagellate morphological identification Charles Kofoid developed a system of plate designation (Fig. 6) that is still used today for morphological identification (Kofoid 1907; Kofoid 1909). The gonyaulacoid and peridinioid cell is divided in reference to various landmarks, the sulcus, cingulum, apex, and antapex. The plates are designated based on their relative position to these landmarks (Kofoid 1907; Kofoid 1909). The plates are marked with a Kofoid label to Figure 6. Peridinioid dinoflagellate diagram depicting Kofoidian plate designation and tabulation designations. The left figure depicts the ventral region and right depicts the dorsal region (O'Toole 2007). designate their position. Armoured dinoflagellates are divided into three zones from the apical to the antapical end of the cell. The epitheca describes structures apical to the cingular girdle (or ?cingulum?). The cingulum is itself a zone, in which lies the transverse flagellum. Those regions of the cell posterior to the cingular girdle are referred to as the hypotheca. The apical series that surround the APC are designated with a number starting with the ventral-most plate of that series being marked 1; the dorsal-most plate would be 8 marked 3, with one diacritical mark, e.g., 3' (Fig. 6). Thus, a number 3 precingular plate, which lies just above the cingulum, is designated by two diacritical marks: 3''. Similarly, postcingular plates that lie just below the cingulum would be 3'''. Plates that make up the antapex region, termed antapical plates, bear four diacritical marks; e.g. 2'''' (cf. Fig. 6). The intercalary plates that lie between both the apicals and the precingulars are designated ?a;? thus 3a. Any intercalary plates that lie between the postcingulars and antapicals plates are designated ?p? (not seen in Fig. 6). Cingulum plates are designated by ?c?. Sulcal plates are defined by their relative positions (left, right, anterior or posterior) and an additional ?s?. a b c Figure 7. Dinflagellates representing the three types of flagellar arrangement a) Peridinium (dinokont) b) Prorocentrum lima (desmokont) (Calkins 2006) c) Oxyrrhis marina (opisthokont) (Calkins 2006) Morphological identification is not based solely on plate tabulations. Although plate tabulations play an important defining characteristic in morphology-based identification, the flagellar arrangement of the mastigote, or swimming cell, is also used to help define dinoflagellate type. Three flagellar arrangements persist among dinoflagellates: dinokont, desmokont, and opisthokont (Fig. 7)(Taylor 1987). In the dinokont arrangement, the transverse flagellum has a ribbon-like appearance, and beats in such a way that it propels water at roughly 90 degrees to the orientation of the cingulum 9 (Leblond and Taylor 1976). The longitudinal flagellum beats posteriorly, propelling the cell forward. In contrast, desmokonts bear both flagella on the anterior end of the cell and are not associated with any grooves. The opisthokonts bear two flagella that arise from the cell posterior end. Girdle displacem ent is also used in the morphological description of dinofla lignment us superclasses Syndinea and Dinoka s is histone proteins in their DNA (Lee, Hutner et al. 1985). A commonality this is shared gellates. Rarely does the cingulum girdle meet up with itself in exact a (Fig. 6). The displacement is referred as either left-handed or descending and right- handed or ascending forming helices around the cell (Fig. 8). In some cases numero turns of the cingulum encircle the cell (Taylor 1987). Infraphylum Dinoflagelleta is divided into two a b c Figure 8. Types of cingular displacement (arrows indicate probable water flow) a) No displacement b) left-handed or descending c) right-handed or ascending (Leblond and Taylor 1976) yota. The morphological separation that distinguishes these two superclasse their nuclear structures. The Dinokaryota possess a dinokaryotic nucleus, a nucleus that contains permanently condensed chromosomes, while the Syndinea do not (Spector 1984a; Cachon and Cachon 1987). However the Dinokaryota do not possess histone associated DNA, (and so are lacking nucleosomes); but instead possess ?histone-like? proteins (Spector 1984c). The Syndinea are differ from Dinokaryota by possessing 10 between these two groups is the presence of an unusual base, 5-hydroxymethyluraci is only found in dinoflagellate DNA (Spector 1984a). l, that Nutritional diversity Dinoflagellates vary nutritionally from being strict autotrophs through heterotrophic organisms. Although strict autotrophs are very rare (Gaines sess some ain ly such sources (Stoecker 1999). Phagotrophic ingestion can involve y ion (food mixotrophic to strictly and Elbr?chter 1987), approximately half of known dinoflagellates pos photosynthetic functionality. The overwhelming majority of photosynthetic dinoflagellates are mixotrophic, facultatively moving from autotrophic to heterotrophic depending on available resources. These photosynthetic species usually cont chlorophylls a, c 2 , and rarely c 1 , ?-carotene. The preferred light-harvesting carotenoid used is peridinin, with a few exceptions using fucoxanthin, which is in turn usual derived from an endosymbiont. Phagocytosis typically mediates the acquisition of other nutritive compounds as vitamins or alternative carbon the consumption of an entire organism, or may involve piercing cells and removing cytoplasmic contents, a process known as myzocytosis (Schnepf and Deichgr?ber 1984). According to Jeong (Jeong 1999), phagotrophy is performed b either engulfment of a prey organism, break down and uptake by pallium format web), or peduncle-mediated uptake of host materials. Engulfment usually involves ingestion at the flagellar grooves or the posterior end of the cell where the entire prey is consumed (Gaines and Elbr?chter 1987). The use of a pallium or food web was first described by (Allman 1855) but it was not until (Odum 1971) that the term saprotrophy was used to describe the feeding mechanism. The dinoflagellate extrudes a delicate 11 10?m P p C H b trophonts. a) Light micrograph of dinoflagellate C a Figure 9. Ectodermal attachment of parasitic attached to ctenophore host b) Ectoparasitic Protoodinium chattonii Hovasse dinoflagellate 1971a). H indicates the host tissue; p indicates the attachment via peduncle (Cachon and Cachon peduncle; and c indicates the cingulum. cytoplasmic net from its thecal pores that digests and absorbs an ensnared prey. After the feeding event the net is retracting back into the cell allowing the cell to search for another food source. The last phagotrophic mechanism is through the use of a specialized organ referred to as a peduncle. The peduncle itself contains longitudinally-arranged microtubules, extensions of the internal microtubular basket (Lee 1977). In some dinoflagellates the peduncle (Fig. 9) is seen as cytoplasmic extension of the protoplasm originating within the epitheca and emerging from the cingular-sulcal interface near the flagellar pores (Spector 1984b). Parasitic dinoflagellates use peduncles to attach to the host, and as a means to collect nutrients from the host. Dinoflagellate symbiosis The nutritional requirements of dinoflagellates are elusive. While some are strict autotrophs, others are heterotrophic; still others are mixotrophic, i.e., capable of both heterotrophy and autotrophy. Many dinoflagellates form symbiotic relationships with 12 other organisms to achieve their nutritional goals. One example of a well-known genus that contains mutualist is Symbiodinium. A group of well-known, closely studied endosymbiotic dinoflagellate that actually represents a very wide range of phylogenetic variation (much more than represents a typical species) (Rowan and Powers 1992), Symbiodinium forms a symbiotic relationship with various corals and other marine organisms. The dinoflagellate is supported by obtaining nitrogen, phosphorus, carbon and other coral metabolites while providing fixed carbon, organic acids, and other various metabolites back to the host (Fitt, Rees et al. 1995; Hackett, Anderson et al. 2004). Some biont and even plastids that are not dinoflagellate in origin. Such plastids are referred to as kleptochloroplasts (Sweeney iting dinoflagellates themselves contain an endosym 1971; Larsen 1988; Fields and Rhodes 1991); that can be photosynthetically active (Skovgaard 1998). Parasitism is a common form of symbiosis for many dinoflagellates. It has even been shown in a laboratory environment that they can shift from mutualist to parasite (Sachs and Wilcox 2006) in the case of horizontal transmission of symbiont to host. It is generally accepted that parasitic relationships evolve from the need for specific, lim metabolites required for survival of the parasite. Usually this concerns organic substrates not available by other means other than the physical removal from a host organism. Most parasitic relationships have very exacting requirements, and involve interactions that display a very limited range of species interactions. However, this is not always the norm with dinoflagellates. An example of multi-host parasitism is seen in Amoebophrya ceratii, a dinoflagellate that can parasitize a multitude of dinophyte species (Drebes 1984). Another example of a dinoflagellate having multiple host is an Oodinium sp. 13 known to parasitize a variety of ctenophores and a hydromedusa (Mills and McLean 1991). However, there are species-specific relationships as in the dinoflagellate Myxodinium pipiens and its host-parasite symbiosis with only Halosphaera. Parasitic dinoflagellates There are more than 2,000 formally described dinoflagellate species, of which approximately 140 are known to be parasitic (Drebes 1984). Dinoflagellates may parasitize organisms extracellularly and/or intracellularly. According to Jean Cachon, parasitic dinoflagellates were categorized into the polyphyletic groups Blastodinida and Duboscquodinida (Cachon 1964). The groups were formed on the basis of morphol nuclear development, and their relationships with the host. Then Loeblich established additional Orders of parasitic dinoflagellates based on biochemical data, the Syndinia and another Order that encompassed members of the genus Chytriodinium and its rela (Loeblich 1982). Today numerous members belonging to the Class Blastodiniph and the Syndiniophyceae, which now contains the Duboscquodinida, have bee ogy, two les tive yceae n molecular data and moved into the Class Dinophyceae, in order to provide phylogenetic relevance to these groups (Coats 1999; Levy, Litaker et al. n rearranged based on recent 2007; G?mez, Moreira et al. 2009; Coats, Kim et al. 2010). The Blastodiniphyceae and some Dinophyceae are known ectoparasites found o or in other protists, or metazoans. The Blastodiniphyceae have a direct physical attachment to the host by a posterior stalk and display a slow morphological change from a free-living form into a parasitic form (Cachon and Cachon 1987). The ectoparasitic dinoflagellate may contain chlorophyll, as seen in Protodinium, Piscinoodinium, and Crepidoodinium, or may entirely lack photosynthesis at any stage of life. This strictly 14 heterotrophic condition is exemplified by Myxodinium, Cachonella, and Amyloodinium species (Coats 1999). If chloroplasts are present they are usually intensely modif their pigmentation can disappear and reappear depending on stages of autotrophy or complete heterotrophy (Cachon and Cachon 1987). Ultrastructure of the ectoparasitic dinoflagellate pedu ied and ncle (Cachon and Cachon 1971a; ht to the ry e stylet can provide support or aid with th e Cachon and Cachon 1971b) shows that it can remain attached to the host surface or penetrate into the host cell in either event forming a network of rhizoids thoug function for uptake of host material. Cachon and Cachon observed the stalk of Protoodinium deeply embedded into its host cytoplasm and believed that it acted as a cytopharynx, a structure acting as a gullet to pass food material from the cytostome to cell interior (Cachon and Cachon 1971a). The peduncle of Amyloodinium has been observed by (Lom and Lawler 1973) to transport small vesicles and organelles from the perinuclear cytoplasm into the host, which was interpreted by Lom as lytic substances used to digest host cellular material. In some dinoflagellates a stylet acts as a seconda structure that works in conjunction with the stalk. Th e removal of host material as noted in the (Lom and Lawler 1973) study on Amyloodinium or Haplozoon (Siebert Jr 1973). An unusual example of attachment is seen in Chytriodinium (Cachon and Cachon 1968) where instead of a peduncle the dinoflagellate uses its hyposome, ventral body, as a spear to penetrate through a crustacean egg, its host; and then later develops a set of organelles to hold itself in plac once it has reached the host cytoplasm. Ectoparasitic dinoflagellates are known to parasitize a variety of gelatinous metazoans. Protoodinium hovassie and Cachonella paradoxa are known parasites of 15 siphonophores, while Protoodinium chattoni is a known parasite of hydromedusae (Cachon and Cachon 1987). A species of Oodinium has been reported to parasitize several gelatinous animals in the Pacific Northwest, including arrow worms, ctenophore and hydromedusae (Mills and McLean 1991). The Duboscquodinida are intracellular and even intranuclear parasites of protists (Cachon 1964; Drebes 1984), with the exception of Sphaeripara, a known metazoan parasite (Chatton 1920; Coats 1999). In general, Duboscquodinida lack theca, chloroplasts, and even mitochondria suggesting that they are indeed obligate intracellular parasites. Gaines and Elbr?chter (Gaines s and Elbr?chter 1987) have stated that parasitic dinofla and . s ge of aped cell possessing a helical girdle (Fritz and Na he apical gellates have ?. . . morphologically different feeding and reproductive stages . . produce . . . numerous progeny after only one feeding act.? The parasitic criterion i very evident in the Duboscquodinida. Between their free-living reproductive phase, sporont stage, and their intracellular parasitic phase, trophont stage, every living sta this group is specialized for the optimization of parasitism. The Amoebophrya cerati sporont is a free swimming biflagellate, pear-sh ss 1992). Amoebophrya experiences an extreme morphological change during the trophont stage (Fig. 10). After infecting its host the cell increases in size, allowing the girdle to elongate and make additional rotations around the cell. The episome, t portion of the cell, sinks into the hyposome, the antapical portion of the cell. Concurrently, the hyposome is enlarged to fold up and over the episome, forming a cavity referred to as a mastigocoel (Cachon 1964). The trophont begins to undergo a growth and division phase during which proliferation of numerous nuclei and flagella are evident. 16 Finally the sporonts exit the host in a tightly coiled multinucleated structured referred to as a vermiform. Figure 10. Diagram of the life cycle of Amoebophrya. a) dinospore; b,c,d) invagination of the growing intracellular trophont (Ma = mastigocoel); e,f) evagination of the trophont phagocytosis of the host and formation of a vermiform; g) lengthening of the vermiform; i,h) formation of the swarmers (Cachon and Cachon 1987). Members of the Order Syndinida are an intranuclear group that parasitize protists and a wide variety of metazoa. Syndinida are dinoflagellates responsible for the decline of many invertebrate (Shields 1994; Appleton and Vickerman 1998; Stentiford and Shields 2005) and vertebrate (Gestal, Novoa et al. 2006) commercial fisheries. Upon the 17 onset of infection into the host hemal sinuses, vascular sinuses lacking a distinct lining and organs during the trophont stage, these parasites convert into a plasmodial form (Shields 1994). The plasmodia are then free to grow and produce a thin polysaccharidic cell coat until sporogenesis of micro and macrospores occur. The ingestion of host material is by performed by sapotrophy (Cachon 1964). Parasitic Groups Characteristics Blastodiniphyceae Ectoparasites of protists, metazoans, and algae. Exhibits a gradual modification of morphology from free-living to parasite. Dinophyceae Endo and ectoparasites of protists and metazoans. Exhibit a broad range of variation. Placement is based on molecular relevance. Possess permanently condensed chromosomes without histones (Spector 1984c). Syndinida Endoparasites of protists and metazoans. Parasites are colorless and from a thin polysaccharide cell coat. May or may not have a theca or cell wall. Chromosomes possess histones (Lee, Hutner et al. 1985) Table 1: Zoological Nomenclature and characteristics of parasitic dinoflagellate groups Parasitic dinoflagellate reproduction The reproduction of parasitic dinoflagellates is based upon three mechanisms presented by (Cachon and Cachon 1987). In Syndinida, cells begin to divide within the plasmo d dial form. After completion, flagellated spores are produced and released. Another mode of reproduction, termed palintomy, occurs after the conclusion of a feeding event. The dinoflagellate, after reaching a substantial increase in size, will begin nuclear an cytoplasmic divisions producing sporonts, termed swarmers. Swarmers are produced when the dinoflagellate becomes multinucleated during a feeding event and can undergo multiple cell divisions either during the feeding event or after. The mechanism, termed 18 iterative sporogenesis or palisporogenesis allows for a single trophont to produce numerous generations of spores (Cachon and Cachon 1987). The production of num generations is accomplished when the divisions of new ce erous lls occur simultaneously with the parasit ew cells can grow in s ivide finally being releas ig. 10). A parasitic symbiont specifically associat ic feeding event. The n ize and then further d ed in a vermiform (F ed with the ctenophore Mnemiopsis apharsodinium there have been no reWithin the dinoflagellate genus Pent ported cases of parasitism. P. tyrrhenicum has been described by (Montresor, Zingone et al. 1993) as a marine benthic autotroph. P. trac associated with e always found been formally described as either autotrophic of heterotrophic. In this study I describe a I dent fication and describe the life cycle of this parasiti hodium and P. dalei, have not been found in benthic samples, although neither have a host and ar specific parasitic relationship between a dinoflagellate and its host Mnemiopsis leidyi. provide a morphological and molecular i i c dinoflagellate. 19 Chapter 2: Collection and Culture of a Dinoflagellate Parasitic on the Ctenophore Mnemiopsis leidyi. Introduction The ability to collect host and parasite has played a very important and limiting role within this study. Availability of the host, mer months. Mnemiopsis and several other ctenophore species collected at multiple sites ranging from Port Aransas and Galveston Bay, Texas; Pascagoula, Mississippi; Mobile Bay at Dauphin Island, Alabama; Pensacola Bay, St. Andrews Bay, Apalachicola Bay, Dickerson Bay and St. Marks Bay, Florida. Bero? ovata were collected in the northern Gulf from Mississippi, and the Chesapeake and Delaware Bays, Bero? cucumis were collected from Pamlico Sound, North Carolina and Woods Hole and Sandwich Harbors, Massachusetts. Pleurobrachia pileus and Euplokamis dunlapae were collected from Cape Cod Bay at Sandwich Harbor and Cape Anne, Massachusetts. Close inspection of all these ctenophores never revealed any dinoflagellates. Mnemiopsis, was critically dependent on several factors, including weather and tide condition. Collection of the host was most successful during high tides. Collection sites known for pristine water quality, such as Apalachicola Bay, produced few dinoflagellates associated with Mnemiopsis. However, sites that appeared hypereutrophic, for example Englehard, NC, Mobile Bay, AL, and Davidson Bay, Florida, provided Mnemiopsis with dense surface loads of the dinoflagellate. Greater numbers of parasitic loads were observed during the spring and sum 20 Objectives and Rationale Objective 1: Establish a viable cell culture of the parasitic dinoflagellate ent of cell cultures allows for the independent study of the parasite ithout ming cell, mastigote. The establishm w the host. Secondarily, collections of freshly caught ctenophores to obtain data on the parasite would not be required. The cultured dinoflagellate could then be used for DNA extraction without interference of host tissue and would also allow for a morphological description of the free-swim Objective 2: Koch?s Postulate The establishment of the cultures also allows for the ability to complete Koch?s Postulates. Specifically, to fulfill Koch?s postulates it would be necessary to: 1) find the microorganism in abundance in all organisms suffering from the disease 2) isolate the microorganism 3) reinfect the microorganism back into the host and show the sam 4) reisolate the microbe and identify it as the original (Koch 1891). e effect Materials and Methods Host collection M. leidyi were collected from locations along the Eastern US and Gulf Coast from shallow water, by wading at near-shore locations, and by boat from estuarine locations and open water sites. At shallow shore sites, ctenophores were collected whenever possible by surface dipping; when obtained via shipboard from the R/V Cape Henlopen or R/V Hugh Sharp (as guest of Dr. Eric Wommack and Dr. Wayne Coats during the MOVE 2007&2008 trips), ctenophores were often collected captured by a very slowly towed 325 ?m mesh plankton net. Approximately 30 animals from each site were thoroughly examined for the presence of microorganisms with the frequency of 1-3 21 collections per month from 2005-2008. Ctenophores were held in 1-2 L plastic jars until arrival at the laboratory. The holding times ranged from a few minutes at the Marine the Marine were observed at 20x? on microscope to assess the protist assemblage. Shipboard-collected ctenophores were observed on site within 1-2 hours upon collection. Biological Laboratory and NERRS/Apalachicola, typically five to eight hours from the Northern Gulf coast locations, and as long as 48 hours from Eastern coastal (Rhode Island to South Carolina, or South Texas) locations. Animals were also obtained from Gulf Specimen Supply, captured from Dickerson Bay in Panacea, Florida, and Biological Laboratory. Upon arrival in the laboratory, the animals 110x magnification by a dissecti Site GPS Site GPS Bayview Ferry, N 37?43.98 NC Station 744 W 76?10.94 Engelhard, NC W 075?59.453 Station 804 W 76?12.76 N 35?30.482 N 38?04.39 Nags Inlet 39?12.74 75?17.06 Head/Oregon JS22(Del Bay) N W N 39?07.52 W 70?20.21 JS28(Del Bay) N 38?48.97 W 74?58.13 Station 908 N 38?57.97 W 76?23.05 Lewes Harbor N 38?47.119 W 75?09.405 Station 858 Station 845 DISL N 38?45.00 W 76?26.00 N 30?15.056 W 88?04.806 Station 834 DISL N 38?34.62 W 76?26.23 N 30?15.158 W 88?04.719 Station 818 N 38?18.01 W 76?16.37 Apalachicola Bay Station 758 Galveston Bay N 37?58.29 W 76?12.61 N 29?17?20.45? W94?52?28.06? N 36?22.97 W 74?26.10 FSU Marine Lab N 29?54?50.74? W84?30?41.19? Off-shore site Station 707 N 37?06.97 W 76?06.93 St. George Island, FL Station 724 N 37?23.94 W 76?04.75 Dickerson Bay Panacea, FL Table 2: Host collection sites and GPS coordinates. During each collection a minimum of thirty animals were collected. 22 Dinoflagellate collection Ctenophores were surveyed for the presence of dinoflagellates after each collection. The density of dinoflagellates on Mnemiopsis varied. Dinoflagellates were removed only from hosts with cell densities ranging from 100 mm -2 to 150 mm -2 (Fig 11). Dinoflagellates were collected from the ctenophore by two methods. Initially, we collected dinoflagellates by braking pipette, or by using a syringe-driven Gilson pipettor. Cells were collected directly by plucking from the surface or by penetration of the mesoglea and targeting specific cysts. This method, while very precise, proved impractical for repeatedly collecting a sufficient number of cells for molecular analysis. . More importantly, cells collected in this manner were not viable for cultivation. Presumably this method of co ged the c d there establishment of viable cultur Subsequently, I devel fficie at e ct larger numbers of cells. Heav ssue by dissection and griseus, Cat. No. 81748 Sigma Chemical llection dama ell an fore impacted es. o eped a more nt method th nabled us to colle ily infested ti s were removed incubated at room temperatur tely 23?C) in 0.1 % (w/v) RNAse and DNAse free protease (type XIV protease, Streptomyces e (approxima O L S L Figure 11 ogra opsis wi dinofl e density. a nd (L . b) (T) ntacular nd partial (S com a b T T . Light Micr ph of Mnemi th high agellate surfac ) (O) Oral a ) subsagittal ) lobe region Subte comb rows a b rows. 23 Co., St. Louis, MO, USA) dissolved in 0.45 ?m filtered ambient seawater. After 1-4 hours t ipetting e he mesoglea collapsed, releasing encysted cells, which were collected by p with a Pasteur pipette. Heavy infestations of ectodermally-attached dinoflagellates wer pipetted directly from the remaining fragments of ctenophore ectoderm and remnant mesoglea. In all cases, cells to be analyzed by molecular techniques were washed in several dishes of sterile filtered artificial seawater then permeabilized, stabilized and fixed by being placed into acetone immediately upon collection. Algal cultures Two methods were used in the collection of algal cells for cell cultures. 1) Attached parasitic cells were removed from the host surface with a braking pipet canted neck flasks (cat. no. 430720, Corning, NY) in K medium (Keller, Selvin 1987), in 30 ppt sterile filtered sea te. Cells were placed into several washes of site 0.45 ?m ? syringe filtered sterile seawater collected from the site of capture. 2) Alternatively ctenophores were placed in Petri dishes containing 0.45 ?m filtered sterile seawater supplemented with full strength K medium (Keller, Selvin et al. 1987) minus silicate, to reduce the likelihood of diatom contamination. Ctenophores (and/or ctenophore tissue fragments) were incubated at room temperature overnight. All algal cells were placed in 96 well culture plates containing fresh K medium. Cells were grown to a high cell density (Fig. 12) at 29 ? 1?C in sterile Corning 75 cm 2 et al. water base collected from St. Andrew?s Bay, Florida. Culture L:D er s were illuminated at an irradiance level of 80 ?mol photons?m -2 ?s -1 on a 12:12 cycle. Cell counts were performed using a hemacytometer (cat. no. 0267110, Fish Scientific, Pittsburgh, PA). 24 The ctenophore parasite was compared with a known strain by culturing. P. tyrrhenicum strain SZN13, a known benthic autotroph, was provided by Monika Kirs Bremen University FRG. SZN13 was originally collected from the Bay of Naples, Italy (Montresor, Zingone et al. 1993). ch, Host reinfection Ctenophores known to be free of dinoflagellates, were collected from the Nati Marine Fisheries jetty by surface dipping in Woods Hole, MA, or from Apalachicola Bay, Florida. After the arrival of Mnemiopsis in the lab, ctenopho onal res were observed each for the presence of dinoflagellates; close inspection indicated that animals of h ng tank and allowed to incubate at room temperature (~23 ?C) for fort day over a week were entirely free of parasitic dinoflagellates. Immediately before infestation, each ctenophore was given a final inspection. Two ctenophores were placed in dinoflagellate-free two liter holding tanks, in each three groups; A, B or C. Group A (control) contained ctenophores not inoculated wit cultured dinoflagellates. Group B consisted of ctenophores inoculated with a single culture of parasitic dinoflagellates isolated from Engelhard, North Carolina. Approximately 35 mL of culture containing approximately 9000 cells mL ?1 were introduced into the holdi y-eight hours. Group C consisted of ctenophores inoculated with a culture of P. tyrrhenicum, strain SZN13 at the same density as in Group B. Ctenophores were observed under a dissecting microscope (model SZ11, Olympus Corp., Center Valley, PA, USA) equipped with an oblique illumination base (model TLB3000, Diagnostic Instruments). 25 Results Dinoflagellate cultures Algal cultures were established and used in the morphological analysis, reinfection experiment, and molecular analysis of the dinoflagellate. When attempting to establish cultures fewer than 50% produced viable cell lines. Single cells grew well when initially inoculated into volumes of less than 100 ?L; larger initial culture volumes did cell lines. Healthy viable cells doubled over a 12 ? 24 hour period. hen c not produce viable W ysts were observed, culture volume was doubled each week until they reached a total volume of 50 ? 75 mL. Cell cultures plateaued at two months (Fig. 12) at approximately 9,000 ? 10,000 cells per mL and then began to decline in numbers. Estimated Parasitic Dinoflagellate Cell Culture Growth 2000 8000 Number of cells per mL Figure 12. The chart represents the estimated growth of isolated inspection estimating by orders of magnitude. Days 60 ? 90 are based cultures. 0 4000 6000 10000 15 days 30 days 60 days 90 days Time parasitic dinoflagellates in culture. Days 15 ? 30 are based on visual on accurate cell counts using a hemacytometer on established 26 Reinfection experiment The first of Koch?s postulates states that the microorganism must be found in abundance when a disease symptom is observed. Ctenophores collected in the field with dinoflagellates resulted in mortality in a time span of less then 12 hrs. to one week. Ctenophores collected that did not harbor dinoflagellates could be keep in holding tanks for several months. The reinfection of the microorganism back to the host after isolation is another criteria to complete Koch?s postulates. Reinfection back to the host utilizing the isolated dinoflagellate yielded approximately 20 ? 30 trophont dinoflagellate cells attached to Mnemiopsis. The attached cells exhibited the typical change in morphological features and growth observed from trophonts on ctenophores captured in the field. However, attachment location was random and did not exhibit the same anatomical Reinfection of host (Mnemiopsis ) 0 100 0 0 10 20 30 40 50 60 70 80 90 100 No dinoflagellates Isolated dinoflagellate added P. tyrrhenicum added Experimental Groups Percent Mortality P. t 27 yrrhenicum Figure 13. Chart representing mortality of host Mnemiopsis in response to reinfection with dinoflagellates. Due to a low number of replicates statistical analysis could not be applied. Parasitic Dinoflagellate preference as seen in low infestation rates of field captured ctenophores. Reinfestation of inoflagellate caused mortality after two days (Fig. 13). Mortali hores ism and ue to the host completely degrading and attempts to locate the dinoflagellate was nonproductive. Also, the low number of trials and sample size, N = 4 per experimental group, does not provide enough data to perform an analysis showing a statistically significant outcome. Discussion and Conclusions the host by the parasitic d ty was assessed by the complete disintegration of the ctenophore. Ctenop that were dying would settle to the bottom of the tank, stop swimming, and begin to deteriorate. Attempts to infect Mnemiopsis with P. tyrrhenicum did not produce any attached dinoflagellates, (Fig 13) and were still alive after 48 hrs. Ctenophores that were not infected with either P. tyrrhenicum or the cultured isolate did not display any mortality within the 48 hrs. time frame. The final component to Koch?s postulates is to reisolate the microorgan establish its identity as the original isolate. However, reisolation was not possible d The collection of ctenophores from various sites was highly dependent upon weather conditions and tidal ranges. Collection trips were planned according to times that high yields were expected. Generally collections of Mnemiopsis yielded over 50 per site but a minimum of 30 was collected to provide a statistically viable collection size. Dinoflagellate collection from hosts using direct physical removal yielded a lower fficiency in establishing viable cultures. It is likely that the forcible removal of the parasite caused damage to the cell leading to a high percentage of mortality. Dinoflagellates that were collected after self-detachment yielded more success in culture e 28 establishment. The growth of cultures depended greatly on the source of the seawater used fo hen The high degree of mortality and similarity of symptoms observed after infection of Mnemiopsis with a pure culture of the isolated parasitic dinoflagellate fulfills Koch?s postulates to identify the dinoflagellate for as the causative agent of Mnemiopsis mortality. However, even though 100% Mnemiopsis mortality was observed, the number of cells that parasitized the ctenophores was low, less than 1% of the dinoflagellate cells introduced. I speculate that the transfer of the dinoflagellates into an artificial seawater medium caused mortality to the cells and or forced many cells to encyst. It is likely that the few cells that were able to reinfect the host were able to quickly locate the host and adapt to a parasitic form before a drop in viability or encystment. The inability for P. tyrrhenicum to form a symbiotic association with Mnemiopsis suggests that the cultured isolate has specific adaptations permitting establishment of a parasitic relationship with Mnemiopsis. Although, this experiment was assessed using a low number of trials and sample size the trend of high mortality over a short period of time relative to infected individuals verses noninfected individuals has been observed in all ctenophores captured. The quick rate of mortality observed in the newly infected ctenophores could have been a compounded effect induced by stress. The typical time before mortality observed r culturing. Seawater that was collected from sites that produced dinoflagellates yielded the best results for achieving a high cell density (Fig. 12). Seawater used from sites that did not yield dinoflagellates or artificial seawater yielded no growth even w using K culture medium. Due to the low rate of growth produced by water other then water collected at sites of high dinoflagellate infestations only water collected from these sites were used in culturing. 29 from field infected ctenophores with low densities of the parasitic dinoflagellate is within seven days but generally greater than two. However, healthy ctenophores that hav placed in small tanks of artificial seawater will only last one to four weeks while health ctenophores held in large tanks > 5 L have been maintained for times exceeding two months. The other ctenophores involved in the experiments all died shortly after a we time period. It is thus my conclusion that the rate of mortality observed in the time frame of 48 hrs. was produced by a cumulative affect of parasite interaction and stress. The parasitic cells that infected th e been y eks e ctenophores did not display the usual attachm at blished data) st ding asitic trophont. However, the introduction of the isolate into an this from occurring. It is there for my conclusion that the ite- ent pattern seen in low densities from field captured ctenophores. Infestations th involve a low density of surface attached dinoflagellates < 100 mm 2 are typically seen on the lobes and oral region (Fig. 11). A behavioral study of Mnemiopsis (unpu shows the host frequently samples the flocculent benthic layer where it is thought the ho first comes into contact with the dinoflagellate, triggering a stimulatory response lea to attachment of the par artificial environment prevents attachment location of the experimental group is not a valid indicator of typical paras host attachment. 30 Chapter 3: Morphology of a Pentapharsodinium species parasitic on the ctenophore Mnemiopsis leidyi. Introduction Morphological characterization has been one of the most important classical methods of taxonomic classification of armored dinoflagellate species. All armored dinoflagellate species may be identified based on the cell cortex plate structure. The Kofoid system, developed by Charles Kofoid (Kofoid 1907; Kofoid 1909) provides the phycologist with a consistent means to morphologically distinguish among a multitude of dinoflagellate genera. Over the years, several improvements have been developed, some resulting in entirely new classification systems (Taylor 1980; Evitt 1985). Barrows and Balech (Barrows 1918; Balech 1980) have noted polarity variation between the epitheca and hypotheca within armored dinoflagellates. They note that variations within the epitheca tend to be conserved and appear to be caused by intrinsic factors. In contrast, variations in the hypotheca appear to be caused by environmental factors. In this study I conducted a thorough Kofodian plate tabulation based on cultured and host-associated cells. I generated a tentative phylogenetic placement based on this tabulation. During the study I also attempted to characterize the mode of cell adherence to the host and establish the nature of the symbiotic relationship between the ctenophore and the dinoflagellate. 31 Objectives and Rationale Objective 1: Obtain an accurate plate tabulation based on the Kofodian system The plate tabulation will allow for genus level placement of the dinoflagellate and reinforce phylogenetic placement based on molecular data. Hypothesis 1: The dinoflagellate should be classified as a species of Pentapharsodinium. Objective 2: Establish the type of symbiotic relationship the symbiont has with Mnemiopsis The establishment of a host-symbiont relationship will allow for the study life cycle and behavior of the dinoflagellate. Hypothesis 2: The dinoflagellate is a parasite strictly associated with the ctenophore into the Mnemiopsi leidyi. Materials and Methods Microscopy Dissections were performed while observing the ctenophore or ctenophore tissue fragments with a dissecting microscope (model SZ11, Olympus Corp., Center Valley, PA, USA) equipped with an oblique illumination base (model TLB3000, Diagnostic Instruments) to pr ovide improved contrast. Dinoflagellates were also examined with a compound microscope by differential interference, phase contrast and fluorescence (model BHS, Olympus Corporation, Tokyo, Japan). Images were collected with color (model Micropublisher 3.3, QImaging Corp., Vancouver, BC, Canada) or monochrome digital CCD cameras (model QICam, Qimaging Corp.). Image optimization and analysis was performed by using Image Pro Plus (Media Cybernetics) or Image J image analysis software. ThumbsPlus software was used for image archiving as well as post capture 32 digital image adjustment where necessary (Cerious Software, Charlotte, NC, USA). Image markup was performed with Macromedia Freehand or Photoshop (Adobe Systems ages of living cells were recorded to videotape with a monochrom e , model 240, Sony Corporation, San Jose, CA, USA), or, in the case of still shots, to an im Int?l. San Jose, CA, USA). Im e Newvicon tube camera (model VE1000, Dage/MTI Corporation, Roeske City, MI, USA) or Sony HyperHAD CCD composite video camera (Sony Corporation, San Jose, CA, USA), and background-subtracted and digitally enhanced with a real-tim image processor (model Argus 10, Hamamatsu, Japan). Images generated by the image processor were saved to S-VHS tape (model SE-180BQ Hitachi Maxell, Ltd. Osaka, Japan), digital 8 tape (digital HandyCam age capture card (Flashbus Spectrim, Integral Technologies, Inc., Indianapolis, IN, USA). Fluorescence microscopy Calcofluor White staining was used to reveal thecal plate boundaries according to the method of Fritz and Triemer (Fritz and Triemer 1985), with minor modifications to account for local salinity. Dinoflagellates were initially fixed in 2 % glutaraldehyde buffered with 0.1 M sodium phosphate made from 0.2 ?m sterile filtered seawater collected at the dinoflagellate collection site and post-fixed in buffered 1 % osmium tetroxide. Calcofluor White M2R (cat. no. F3543, Sigma Chemical Corp) was added to a final concentration of 10-20 ?g/mL and the cells viewed under UV fluorescence using a Hoechst Ploem cube (model 11000, Chroma Tech, Brattleboro, VT, USA; Olympus BHS icroscope, Tokyo, Japan). The fluorescence images provided the basis for a Kofoidian ber of thecal plates, their morphology and their relative ent. m plate tabulation, based on the num arrangem 33 Scanning Electron Microscopy and Transmission Electron Microscopy visuali te at room temperature for r a 0 minutes prior to use; the sample was cooled on ice for a few minutes in 80mM sodium cacodylate and 0.2 ?m ur. Samples were subsequently washed 3X using ice-cold 80mM m filtered site water and then allowed to incubate for 5-10 fixation osmication was carried out at 0?C using 1% osmium tetroxide in 0.2 ?m in ), On several samples a membrane stripping technique was utilized in order to ze the thecal plates. Cells were placed into a 0.1% Triton X solution mixed in site 0.2 ?m filtered site-collected seawater and allowed to incuba ten minutes. Fixation for electron microscopy was carried out using a ?simultaneous fixation? method (Tamm and Tamm 1981). All reagents were cooled to 0?C on ice fo minimum of 3 prior to the initial fixation. The primary fixation consisted of 1% paraformaldehyde, 2.5% glutaraldehyde, 1% osmium tetroxide buffered filtered site water. Samples were fixed on ice for a minimum of 30 minutes to a maximum of one ho sodium cacodylate in 0.2 ? minutes. Post filtered site water. Samples were incubated for a minimum of 15 minutes to a maximum of 30 minutes in OsO 4 seawater. Samples were then washed 3X in ultra pure water at room temperature. Samples prepped for TEM were stained en bloc overnight saturated aqueous uranyl acetate. TEM and SEM samples were both then subjected to a graded ethanol dehydration series, 15%, 30%, 50%, 70%, 90%, 95% (2x), 100% (3x and anhydrous (2x). SEM samples were treated with 3x exchange of hexamethyl- disilazane (CAS # 999-97-3, Electron Microscopy Sciences). The sample was left overnight in the final exchange of hexamethyldisilazane and then collected for viewing on Zeiss EVO 50 after the chemical had completely evaporated. TEM samples were infiltrated and embedded into Spurr?s resin (Spurr 1969) then sectioned for viewing on 34 Zeiss EM 10C 10CR Transmission Electron Microscope. All reagents and supplies fo the preparation were obtained from Electron Microscopy Sciences (Warrington, PA). Results r Apical Plate Morphology The free swimming mastigote was approximately 25-30 ?m long and 20-25 ?m wide, thereby presenting a pear shape cell body that exhibited dorso-ventral compression, a purely peridinioid characteristic (Fig. 6). It had a dinokont flagellar arrangement; i.e., both flagella were inserted on the ventral side of the cell. The thecal plates had a grain or ?pustulate? appearance (Williams, Sarjeant et al. 1978) and were covered with trichocyst pores placed in irregular patterns, with the exception of a concentric ring y, Figure 14. SEM of parasitic dinoflagellate showing surface ornamentations. The surface is covered in open pores, a texture referred to as pustulate. Note ring of pores immediately above and below the cingulum girdles. above and below the cingulum girdles (Fig. 14). The plate tabulation formula matches that for Pentapharsodinium Po, X, 4?, 3a, 7?? 4C + T, 4S, 5???, and 2???? as proposed by Balech?s description of Peridinium tyrrhenicum n. sp. (Balech 1990). The 1? apical plate represents an ortho conformation, bordered by four apical plates (Fig. 15). 35 Figure 15. 1000X Light micrograph of parasitic dinoflagellate showing ortho conformation. The 1' plate is bordered by the four plates 7'', 4', 2', and 1'' giving the epitheca, anterior portion of the cell, an ortho conformation. The 2a intercalary plate depicts a hexa or six sided conformation. On the basis of these plates it is assumed the epitheca is ortho-hexa. The epitheca appears conical without the presence of apical horns. Indelicato and Loeblich stress that the suture positions of Figure 16. Calcofluor White staining under UV fluorescence. The parasitic dinoflagellate shows hexa conformation. The 2a intercalary plate is shown bordered by six plates 3'', 4'', 5'', 1a, 3', 3a giving the epitheca, anterior portion of the cell, a hexa conformation. 36 the cingular and hypothecal plates are a conserved feature within the peridinioid corticotype, which makes them useful tools in morphological identification (Indelicato lich 1986). and Loeb Cingulum Morphology The cingulum is composed of 5 cingular plates (4C + T) and is displaced, descending from the proximal end. As part of the cingulum description, the transitional plate is designated as the T-plate, with the next attached cingulum plate being the 1C plate. The homologous cingular suture Y found in peridinioids (Indelicato and Loeblich 1986), lies between plates 1C/2C, apical to the 1/2 postcingular suture (Fig. 17a). addition, the X suture lies between plates 3C/4C found apical to the 4/5 postcingular In suture (Fig. 17b). This is the situation in genus Pentapharsodinium. e 2C/3C 1c 2c 1'' 2''' 5''4'' 4c 3c 3 2c 3c 3'' a b c Figure 17. Calcofluor White staining under UV fluorescence depicting shows the X suture. c) The arrow shows the relative position of the 2C/3C suture homologous cingular sutures. a) The arrow shows the Y suture. b) The arrow to the 3 postcingular plate. Another feature consistent with Indelicato and Loeblich?s description of the Pentapharsodinium cingular sutures is the dorsal suture position that form th 37 border positioned at the center of the 3 postcingular plate (Fig. 17c) (Indelicato and Loeblich 1986). Antapical and Sulcal Morphology The antapical plate 1 is approximately one quarter the size of the antapical plate 2 (Fig. 18a), which in turn spans over the majority of the posterior region. The antapical 1 '' 2 ''' 3''' 1 '''' 2 '''' 3''' 2''' 1''' 12'''' 4''' 5''' a b the central region of the 2''' plate. '''' Figure 18. Calcofluor White staining under UV fluorescence depicting antapical plate morphology. a) Shows the asymmetry in the 2''' plate. b) Shows the relative size difference between the 1'''' and 2'''' plates and the border of the plates placed in 2?? 1?? 1?? 5?? 4?? 2?? 3?? a b c atpharsodinium trachodium and c)Pentapharsodinium Figure 19. Diagram a) from (Balech 1990) depicting antapical plate morphology of Pentapharsodinium tyrrhenicum. b/c) (Indelicato and Loeblich 1986) depicting the antapical plate morphology and X and Y sutures of b) Pen dalei 38 plate 1 causes asymmetry of the postcingular plate 2 (Fig. 18b), creating a shorter posterior margin than is seen on the right margin. Four plates cha left racterize the sulcal region, ventral portion of the cell where the l: sa, anterior sulcal plate; sp, posterior sulcal plate; ss, left sulcal p itudinal of e 7 on the right. The left border is shared approximately half way between the right border of the ss and the right border of the sp plates. The sd plate terminates on the posterior end forming a border with the anterior side portion of the sp plate. The right side of the sd plate forms the left borders of the postcingular 5 and cingular 4 plates. The ss plate forms an anterior border against the flagellar pore and the ventral midsection of the transitional plate. The left side forms the right border of the postcingular plate 1 and the left corner may or may not touch the antapical plate 1. The anterior border of the ss plate slightly protrudes into the sp plate forming its anterior border. The right forms a border with the mid to anterior left portion of the sd plate. The sp plate is approximately two times longer than it is wide. The ss plate concaves the anterior border. The left is bordered by the antapical plate 1. ht flagella insert into the cel late; and sd, right sulcal plate (Fig. 20). The sa plate borders upon the long flagellar pore and also forms the right border of the transitional plate. The anterior portion of the sa plate forms the ventral border of the apical plate 1and the left border the precingular plate 7. The posterior end of the sa plate forms the anterior border of the sd plate. The sa plate is somewhat quadrangular or pentangular and is more long than narrow. The sd plate is approximately 3x longer than it is wide. The anterior border is shared by the sa plate on the left and a portion of the precingular plat The posterior forms a border with mid-anterior portion of the antapical plate 2. The rig 39 posterior border forms the right posterior border of the postcingular plate 5. The righ anterior portion i t s bordered by the sd plate. Figure 20. Calcofluor White staining under UV fluorescence depicting sulcal region. The sd represents the right sulcal plate; sa, anterior sulcal plate; sp, posterior sulcal plate; and ss, left sulcal plate. Life cycle and association with the ctenophore host Nonencysted parasitic dinoflagellates attached to Mnemiopsis range from approximately 19-26 ?m long and 15-22 ?m wide with a hyaline layer (Fig. 21b). Increased size variation has been observed in dinoflagellates attached over periods of time greater than three days. Such cells vary in size from 90-300 ?m. Cysts present in Mnemiopsis vary from approximately 25 to 32?m in diameter and are always found embedded within the mesoglea (Fig. 21c). Concentrations of dinoflagellates on Mnemiopsis collected on the East Coast (U.S.) increase during the Spring and Summer months, usually reaching surface densities greater than 150 mm 2 . In contrast, animals collected during the Winter and Fall months usually have very few to no dinoflagellates. 40 41 10 ?m 300 ?m 10 ?m a b c icting ) Figure 21. Dinoflagellate cells associated with host. a) Light micrographs dep high dinoflagellate cell density on a ctenophore captured during the Spring. b Dinoflagellate attached to host via peduncle (P). c) Encysted dinoflagellate cell embedded into host tissue. P a b d e f c P C P CCC P P C C N P Figure 22. Light and SEM micrographs of dinoflagellate peduncle (P) and hypotheca (C) a) 1000X showing peduncle and numerous fimbre (arrow). b) SEM of unattached peduncle. c) Light micrograph of dinoflagellate skipping across host tissue. unattached peduncle (arrow) and nucleous (N) d-f) Light micrographs showing a focal series of unattached dinoflagellate peduncle. The dinoflagellate attaches via a peduncle penetrating into the host ctenophore? epidermis (Fig. 22a). The dinoflagellate was s usually found in greater abundance in the auricular grooves and the oral region, as previously described (Moss, Estes et al. 2001). a b c d e N f PP hg PP Figure 23. Light micrograph series showing parasitic reproduction, termed palintomy after disassociation with host. a) Shows single trophont as uninucleated (N). b) Detached cell undergoing equal holoblastic cleavage ( stage). c-d) Detached cell undergoing a the 4-cell divison stage. e-f) Detach undergoing 8-cell stage the peduncle (P) can be seen and the outer cell membrane (arrow). g-h) Probable 16-cell stage multiple new dinospores are present (arrow). 2-cell ed cell 42 Video-DIC micrography of detached dinoflagellates next to epidermal fragments revealed that it moved from place to place upon the host tissue until a location was found for attachment (Fig. 22c). Attachment occurred very quickly, in less than a minute. Cytoplasmic streaming, possibly of membrane-bounded vesicles containing lytic enzymes, was immediately and clearly observed undergoing orthograde transport within the peduncle. After a few minutes what is believed to be degraded host material was visualized to stream up the peduncle into the cell. arasitic Reproduction (the tomont)P The dinoflagellate associated with Mnemiopsis undergoes an unusual form of reproduction only seen in parasitic dinoflagellates: palintomy. The attached trophont enters a growth phase after attachment to the host. Cells that have been attached to the host for longer than three days have been observed to be as large as 100 ? 300 ?m in size. It is thought that after the feeding event is over the dinoflagellate enters into its reproductive phase, the tomont (fig 23 b-h). The cell begins to undergo division within the cell membrane and then ruptures, releasing a multitude of dinospores or sporonts or swarmers. Swarmers are produced by all parasitic dinoflagellates and are there for a good indicator of a parasitic lifestyle (Cachon and Cachon 1987). Swarmers possess two flagella, have a poorly developed girdle and sulcus, are morphologically variable compared to the Mastigote, and may be produced in macro or micro forms (Cachon and Cachon 1987). In the lab I have observed numerous tomonts rupture and produce inospores that moved along the host tissue and appeared to possibly attach (Fig. 24) or swim away. d 43 T S T S T T Figure 24. In situ SEM micrographs depicting putative dinoflagellate sporonts, swarmers and trophonts. a) Several different size trophonts (?T?) appear, attached to host tissue. Thin arrow shows a sporont. b) Sporonts (?S?) cin 44 Discussion and Conclusions Morphological analysis based on Kofoid?s plate tabulation scheme places the dinoflagellate within the genus Pentapharsodinium. However, there are some discrepancies in the literature supporting the validity of the current plate tabulation designated for the Pentapharsodinium genus Po, X, 4?, 3a, 7?? 4C + T, 4S, 5???, and 2????. A dinoflagellate belonging to the genus Ensiculifera based on molecular characterization of SSU, coupled with ITS analysis, has been noted to possess the same plate tabulation as the genus Pentapharsodinum (Hai-Feng and Yan 2007). The current plate tabulation used in the morphological identification of Ensiculifera is Po, X, 4?, 3a, 7?? 4C + T, 5S, 5???, and 2????, (Matsuoka, Kobayashi et al. 1990) note the number of sulcal plates is 5S rather than 4S. Also, another morphologically distinct character of Ensiculifera is the presence of a long slender spine, about half the length of the epitheca, arising from the right anterior corner of the T plate (Fig. 25). The spine present in Ensiculifera is not without scrutiny as well. ITS analysis by the D?Onofrio group (D'Onofrio, Marino et al. 1999) could not separate Ensiculifera as an independent genus from Pentapharsodinium even gular girdle is indicated by a triangular arrow, while a boxed arrow shows a flagellum. Note tenfold difference in scales. b a after coupled with a morphological anlaysis. D?Onofrio criticized the validity of th presence or absence of the spine as a valid taxonomic character at the genus level. The epitheca of the parasitic dinoflagellate has an ortho-hexa conformation w no horns; both are indicative of the peridinioid group. The dinoflagllate adheres to complete plate tabulation set for Pentapharsodinium Po, X, 4?, 3a, 7?? 4C + T, 4S, 5???, e ith the n the a defining characteristic for the genus Pentapharsodinium. The antapical plate size, shape, and general structure relative to the known Pentapharsodinium species Figure 25. Diagram depicting the sulcal region of an Ensiculifera species (Matsuoka, Kobayashi et al. 1990). Note the large spine associated with the T plate and the presence of 5 sulcal plates. and 2????. The Y and X sutures of peridinioids specifically fall within the descriptio placed on the Pentapharsodinium genus. Another feature that is purely a Pentapharsodinium characteristic is the dorsal cingular suture position that forms 2C/3C border positioned at the center of the postcingular plate 3. The antapical plate morphology is 45 (Fig. 19) suggest that this dinoflagellate could possibly be an undescribed species. It is possible that the variation found in the antapical region is a result of the dinoflagellate adjusting to environmental changes as indicated by (Barrows 1918; Balech 1980) that caused it to adapt to a parasitic lifestyle. Another more likely possibility is that the antapical variation is a result of the dinoflagellates adaptation to parasitism. Due to uncertainty in the literature involving Pentapharsodinium and Ensiculifera, in part due to their close genetic relationship and the high degree of morphological similarity, it is inconclusive whether if the dinoflagellate in this study elongs in the Pentapharsodinium genus. Given the current accepted plate tabulation of Pentapharsodinium, I tentatively place the new dinoflagellate into the Pentapharsodinium genus. Evidence for parasitism b The peduncle appears to penetrate host epithelial cells, thereby facilitating of nesis myzocytosis. SEM (Fig. 22b) and video (not shown) all suggest that the peduncle is hollow, and possibly lined with microtubules and F-actin that transport lytic enzymes to penetrated host cells and recover digested host material back to the cell. Cell adherence occurs very rapidly, in less than one minute, and the transport of materials occurs almost immediately upon adherence. Cells that are observed attached to the host over a period days grew very rapidly in size, some exceeding 200?M. Sporogenesis of this parasite is different from the description given by Cachon and Cachon for palintomy in Protoodinium chattoni Hovasse (Cachon and Cachon 1987). In P. chattoni, sporoge occurred after a feeding event was completed and the dinoflagellate detached from the 46 host. However, in this study, sporogenesis was observed while the cell was still attach to the host. ed preparation) revealed that the host frequently samples the flocculent benthic layer. Our operating hypothesis is that the ctenophore may recruit the dinoflagellate from currents generated as the ctenophore rests against the substrate. The heaviest initial infestations of mesogleal cysts occur in the stomodeal walls and in the tissues underlying the auricular grooves. Trophonts, sporonts and swarmers were observed in their greatest numbers after observation of mesogleal cysts, which were observed to migrate very slowly through the mesoglea after infestation, presumably via the feeding apparatus, to locations on the host surface. Host specificity I propose that the host becomes infected by dinoflagellates because of its interaction with specific sediments that bear the dinoflagellate as a benthic form. A behavioral study of Mnemiopsis (Moss, Taylor, Odom, Stephenson and Welch, in Mnemiopsis was never systematically examined for transfer of the dinoflagellate to ctenophore predators such as the ctenophore Bero? ovata or the schyphomedusa Chrysaora quinquecirrha. However, even though each individual Bero? and Chrysaora d n any fish certainly collect many hundreds of Mnemiopsis during their life span, I never observe the dinoflagellate associated with either species; nor was it ever evident o known to ingest Mnemiopsis, such as Petrilus burti or Menidia beryllina. 47 Chapter 4: Pentapharsodinium Molecular Analysis Introduction The phylogenetic placement of an unknown organism can be ascertained throug the relative comparison of phylogenetically conserved nucleic acid sequences in dif organisms. Molecular phylogenetic analyses have enabled an additional, obje h ferent ctive ethod gy. In . The wn species and mine the molecular phylogeny of a previously unknown parasiti e m for the classification of organisms, in addition to analyses of morpholo 1991 a new taxon, the Alveolates, was established by virtue of molecular analyses Alveolates are comprised of ciliates, apicomplexans, protoalveolates and the dinoflagellates (Gajadhar, Marquardt et al. 1991; Wolters 1991). Infraphylum Dinoflagelleta is diverse , and is currently comprised of over 2,000 kno 125 genera (Drebes, 1984). In this study I deter c dinoflagellate of the ctenophore Mnemiopsis leidyi. This study includes th analysis of three nuclear gene regions: 1) the (18S) ribosomal small subunit ; 2) the internal transcribed spacer region between the 18Sand 5.8S ribosomal DNA; i.e. ITS 1, and the 3) the ITS2 region, which lies between the 5.8S and the 28S regions. Finally, I present my results on 4) the extranuclear, mitochondrial gene cytochrome b. The sequences were used to construct phylogenetic trees, comparing the sequences with those of other organisms including known dinoflagellates. Analysis of each of the selected gene regions has a particular role in th development of the phylogeny of organisms. The 18S rDNA gene sits within the eukaryotic ribosomal operon. The 18S rDNA encodes for 18S rRNA that is used as a e scaffold for proteins to construct the 40S (small subunit) of the ribosome. Due to the 48 importance of the 18S rDNA gene insertions, deletions, or point mutations that would prevent the assemblage of the 40S subunit are selected against providing the gene with a conserved nucleotide sequence. Due to this level of nucleotide conservation, 18S rDNA ly dergo significantly different origin, like those for cytochrome b and 18S rDNA have hylogenetic trees within the alveolates (Rathore, Wahl et al. 2001). Object is typically used to resolve to the genus level. Intronic sequences like the ITS are on restricted by structure and are under very little selection pressure. The ability to un genetic drift without causing detrimental affects to the cell allows the ITS region to be used in phylogenetics to resolve different populations within a species. Cytochrome b is a mitochondrial gene used in the electron transport respiratory chain for the production of ATP. Mitochondrial genes display a higher rate of change with time, than is seen for nuclear genes (Brown, George et al. 1979). This allows the investigator to use mitochondrial genes to resolve differences at the population level (Conway, Fanello et al. 2000). Analyses that involve coupling sequencing of multiple genes of provided robust p ives and Hypotheses Objective 1: Conduct a multi-locus phylogenetic analysis Sequencing of targeted genetic regions will provide sufficient molecular sequence data to conduct a phylogenetic analysis for the parasitic dinoflagellate and related species. Hypothesis 1: The dinoflagellate should be molecularly classified as a speci es of Pentapharsodinium. 49 Materials and Methods DNA extraction 20-150 acetone-fixed parasitic dinoflagellates, derived directly from the host or from culture, or a similar number of cultured SZN13 cells, were centrifuged at 10,000g in a benchtop microcentrifuge (5415, Eppendorf, Federal Republic of Germany) for 10 minutes at 23 ?C. Pelleted cells were extracted by the Cetyl Trimethyl Ammonium Bromide (CTAB) method modified after Gast et al., (Gast, Dennett et al. 2004) as modified from (Kuske, Banton et al. 1998). Polymerase Chain Reaction (PCR) and sequencing The 18S rDNA gene and the internal transcribed spacer (ITS) regions of the nuclear genome were amplified via PCR with primer pairs Dino18S5F1/Dino18S5R1, airs ied 8.3), 4 2?C, pair (Rowan and Powers 1991) also ation at (Zhang, Bhattacharya et al. 2005) and the internal transcribed spacer region primer p ITS1/ITS4 (White, Bruns et al. 1990). The polymerase chain reaction (PCR) was carr out by incubating 50ng of template DNA with 10 ?M primers, 10 mM Tris-HCl (pH 50 mM KCl, 200 ?M dNTPs, 1 U Taq polymerase and 2.5 mM MgCl 2 in a total volume of 25 ?L. PCR was carried out as follows: an initial 60 s preheat at 94?C, followed by 3 cycles of 45 s denaturation at 94?C, 45 s annealing at 50 ?C, 1 min elongation at 7 and a final period of elongation for 300 s at 72?C. PCR amplification with the SS5/SS3Z primer used approximately 50 ng of template DNA, but instead began with 90 s denatur period at 94?C, followed by 30 cycles of 60 s denaturation at 94?C, 60 s annealing 56?C, 90 s elongation at 72?C, with a final elongation of 5 min at 72?C. The 50 amplification of mitochondrial primers Dinocob1F/Dinocob1R (Zhang, Bhattacharya e al. 2005) were performed under the same conditions as specified in that study. The primer pairs 633DinoF/1051DinoR, D946F/D1582R, and D400F/D965R were developed during this study to obtain the internal nucleotide sequences across the 18S rDNA gene. After consensus sequences were obtained of the 18S rDNA flanking regions using the previously mentioned primers, new primers were built, using Amplify 3X (University of Wisconsin Ver. 3.1.4, Madison, WI, USA) to generate t overlapping regions across the 18S rDNA gene in order to subsequently build contiguous sequences. . t da n ilter. Images were acquired by video capture (model LG3 image analysis performed f the esearch Instrumentation Facility on an ABI 33100 sequencer. The ences ne Codes Corporation Ver. 4.8, Ann Arbor, MI, USA). The consensus sequences were then organized to form contiguous sequences. PCR was carried out under the same conditions as stated for Dino18S5F1/Dino18S5R1 The resulting amplicons were assayed by 1% agarose gel/TAE electrophoresis a 95 V for 35 minutes at room temperature in a Horizon 58 gel apparatus (Gibco/Bethes Research Laboratories, Bethesda, MD, USA). Gels were stained with 0.1 % ethidium bromide. The gel was photographed with a high performance CCD camera (Cohu Inc., San Diego, CA, USA) equipped with a 4-48 mm zoom television lens equipped with a ethidium bromide ?rainbow? f capture board, Scion Corporation, Frederick, MD, USA), and by Gel-Pro software (Media Cybernetics, Bethesda, MD, USA). Successful amplicons were subjected to dye-termination sequencing at the Genetic Analysis Laboratory o Auburn R chromatograms were visualized, edited and assembled to produce consensus sequ using Sequencher (Ge 51 Primer Sequence Region Specificity Reference Dino18S5F1 5?-A GTT AG GGT TGT TAT TAG NTA 18S rDNA 194-220 P. tyrrhenicum ribosomal operon Zhang et al., 2005 CAG AAC-3? Dino18SR1 5?-GAG CCA GATR 18s 683 - 665 Zhang et CWCA CCC AG-3? rDNA P. tyrrhenicum ribosomal operon al., 2005 SS5 (F) TGC CAG TAG TCA an ers 5?-GGT TGA TCC TAT GCT TG-3? 18S rDNA 6-34 P. tyrrhenicum ribosomal operon Row and Pow 1991 SS3Z (R) CAG TCC GAA TAA 18S rDNA P. tyrrhenic 5?-GCA CTG CGT TTC ACC GG-3? 1686-1657 um ribosomal operon Rowan and Powers 1991 633DinoF AGG ACG ACC 18S rDNA 633-657 Internal to contiguous sequence This study 5'-GGA TTT CGT GGT CCG C-3' 1051DinoR 5'-CCT CCA ATC TCT AGT CGG CAT GG-3' rDNA Internal to contiguous sequence study 18S 1051-1029 This D946F GAT GTT TTC ATT 5'-TTT GCC AAG GAT-3' 18S rDNA 946-969 Internal to contiguous sequence This study D1582R 5'-CTG ATG ACT CGC GCT TAC TAG GAA-3' 18S 1582-1559 This rDNA Internal to contiguous sequence study D400F CAC ATC TAA GGA 18S rDNA Interna 5'-AAC GGC TAC A-3' 400-421 l to contiguous sequence This study D965R TCC TTG GCA AA- 5'-ATG AAA ACA 3' 18S rDNA 965-946 Internal to contiguous sequence This study Table 3. List of 18S rDNA primers used in this study, their specificity and referen sources. ce 52 Primer Sequence Region Specificity Reference ITS1 (F) hite et 5'-TCC GTA GGT ITS 1770-1788 P. tyrrhenicum W GAA CCT GCG G-3' 1&2 ribosomal operon al.1990 ITS4 (R) TAT TGA TAT GC- ITS 1&2 2428 ? 2409 P. tyrrhenicum ribosomal operon White et al.1990 5'-TCC TCC GCT 3' references. Primer Sequence Region Specificity Reference Table 4. List of ITS primers used in this study along with their specificity and Dinocob1F CAT TTA CAW WCA TAT CCT TGT CC-3' cyt b 61-92 P. piscicida cyt b operon Zhang et al., 2005 5'-ATG AAA TCT Dinocob1R GKA ATT GWK cyt b 877-850 P. piscicida cyt b operon Zhang et al., 2005 5'-TCT CTT GAG MAC CTA TCCA-3' and references. Sequence alignment and tree assembly Table 5. List of cytochrome b primers used in this study along with their specificity Dinoflagellate 18S, ITS, and cyt b sequences were obtained from GenBank to perform a phylogenetic analysis of the dinoflagellate in this study. Contiguous sequences and sequences obtained from GenBank were aligned using ClustalX ver. 2.0.10 set on Multiple Alignment Mode (Larkin, Blackshields et al. 2007). Aligned sequences were then entered into RAxML (Randomized Axelerated Maximum Likelihood) using a GTR (Generalized Time Reversible) set to 1,000 bootstrap replicates to produce a maximum likelihood phylogeny estimation based on nucleotide sequences (Stamatakis, Hovver et al. 2008). Output files were converted to phylogenetic trees using TreeView (Page 1996). 53 xML is a or seq and paralle Likelihood e the distributio es the obser the greatest probability. ML is essentially an estimation that searches over all possible outcom ase GTR, to most likely scenario, a phylogenetic tree, based on the given data, the sequence alignments (Frongillo 2002). The GT study gives RAxML the parameter values used in its estim to a s. The GTR model was developed by Simon Tavar? (Tavar? 1986) when attempti e t e of numerous Markov models of DNA sequence evolution. In these models a set p re su o ls er assume that nucleotide changes occur at equal frequencies ? the JC and K2P models ? or r 5, R models. Specifically, in the GTR model, Tavar? takes into account the relative roles of substitution, insertion and deletion, duplication, and transposition as forces that change th l assumes th changes are reversible.? In other words, if a nucleotide changes, it has the ability to revert back to the ori cl odel assum ty between nucleotides occurs at different rates and that each nucleotide can occur at dif equ 9 RA program f uential l Maximum based inferences. Maximum Likelihood ref rs to n that giv ved data es giving a specific model, in this c produce the R model used in this find the best phylogenation etic tree with the given data, the sequence lignment ng to and is on of xplain subs itution rates in his study on the divergence time of rat and mouse; arameters a given based on the bstituti n rates of nucleotides. The mode can eith that the four nucleotides can change at diffe ent frequencies ? the F84, HKY8 and GT e structure of genes over time. The mode at nucleotide ?time ginal nu eotide at the same rate. The m es substitution that each pe ferent fr encies (Hillis, Moritz et al. 1 96). 54 Gen Bank No. AF022153.1 2 .1 AF274265.1 AF022198.1 AF022201.1 AB185114.1 AF0 AB088315.1 AB088333.1 AF022195.1 AY800 2154 130.1 AF033865.1 EF492487.1 HQ845328.1 (this study) AF274270.1 AY443018.1 U52357.1 GenB accession analysis BPs 1752 1744 1801 1800 1803 1799 1801 1753 1801 1778 1797 1569 1755 1803 1751 1755 1802 Family Ceratiaceae Ceratocoryaceae Gonyaulacaceae Gonyaulacaceae Gymnodiniaceae Kareniaceae Heterocapsaceae Heterocapsaceae Heterocapsaceae Ostreopsidaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Order Gonyaulacale s Gony cale aula s Gonyaulacale s Gonyaulacale s Gymnodiniale s Gy mnodiniale s Peridiniales dini Peri ales Peri ales Pe es Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales dini Peridiniales ridinial Table 6a: List of dinoflagellate species and Dinoflagellate species ank nu mb er for S SU Heterocapsa niei strain CCMP 447 Heterocapsa triquetra Coolia monotis isolate CCMP1345 Ensiculifera aff. loeblichii strain GeoB*220 Pentapharsodinium sp. Pentapharsodinium sp. CCMP771 Pentapharsodinium tyrrhenicum Peridinium cinctum Peridinium wierzejskii Scrippsiella nutricula Ceratium fusus Ceratocorys horricda Alexandrium fraterculus Alexandrium tamarense Gymnodinium mikimotoi Takayama cf. pulchellum Cachonina hallii 55 Gen Bank No. 3 3.1 AB183677.1 AF274276.1 AF244277.1 AY033487.1 AY456118.1 AY121856.1 AY443022.1 EF492511.1 EU287485.1 AF022156.1 AY051087.1 AY051093.1 HQ324899.1 FJ217814.1 AY434687.1 HM483399.1 DQ 6705 BPs 1756 1755 1753 1787 1752 1792 1751 1795 1764 1792 1531 1531 1740 1521 1798 1801 1705 Family Peridiniaceae Peridiniaceae Peridiniaceae Pfiesteriaceae Pfiesteriaceae Pfiesteriaceae Protoperidiniaceae Prorocentraceae Prorocentraceae Pyrocystaceae Symbiodiniaceae Symbiodiniaceae unclassified unclassified unclassified unclassified Thraustochytriidae Order Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Prorocentrales Prorocentrales Pyrocystales Suessiales Suessiales unclassified unclassified unclassified unclassified Labyrinthulida Table 6b: List of dinoflagellate species a Dinoflagellate species nd GenBank accession number for SSU analysis Scrippsiella sp. MBIC11168 Scrippsiella sweeneyae strain CCCM 280 Scrippsiella trochoidea strain CCCM 602 Pfiesteria-like Pfiesteria-like sp. CCMP 1827 Pfiesteria-like sp. clone POC-8 Protoperidinium pellucidum Prorocentrum micans isolate UTEX 1003 Prorocentrum mixicanum strain CCMP 687 Pyrocystis noctiluca Symbiodinium sp. AP310 Symbiodinium sp. K192 Azadinium poporum isolate UTHD4 Azadinium spinosum strain 3D9 Dinophyceae sp. W5-1 Duboscquodinium collinii isolate VSM11 raus chyt ium nnei Th to r ki (outgroup) 56 Gen Bank No. AB192301.1 AB374987.1 AB355141.1 AB436946.1 AJ312277.1 AB233377.1 AB436947.1 EF036540.1 AB084094.1 AB084095.1 AB445394.1 AB084100.1 AB084101.1 AY728076.1 AY499513.1 AF527814.1 (this study) BPs 578 590 590 584 520 608 605 3264 590 597 568 588 594 555 583 551 593 Family Dinophysiaceae Dinophysiaceae Dinophysiaceae Gonyaulacaceae Gonyaulacaceae Gonyaulacaceae Gonyaulacaceae Gymnodiniaceae Heterocapsaceae Heterocapsaceae Heterocapsaceae Heterocapsaceae Heterocapsaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Order Din Din Din Gon Go Go ophysiales ophysiales ophysiales yaulacales nyaulacales nyaulacales Gonyaulacales Gymnodiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Table 7a: List of dinoflagellate species and GenBank accession number for ITS analysis Dinoflagellate species Dinophysis acuminata Dinophysis infundibulus Dinophysis rotundata Alexandrium fraterculus Alexandrium minutum Alexandrium tamarense Alexandrium tamiyavanichi Karlodinium micrum Heteocapsa pygmaea Heterocapsa arctica Heterocapsa sp M-2008 Heterocapsa sp NIES-473 Heterocapsa triquetra Ensiclifera aff imariensis isolate D207 Ensiculifera aff loeblichii GeoB 229 Ensiculifera cf imariensis strain JB3 Pentapharsodinium sp. 57 Gen Bank No. AF527817.1 AY499512.1 EF417297.1 EU445322.1 AB232669.1 DQ344043.1 DQ344035.1 AY590478.1 DQ238042.1 EU244475.1 DQ238043.1 EU244467.1 EU244473.1 EU786090.1 FJ626951.1 AF316893.1 BPs 554 586 558 570 3602 677 772 3320 3208 631 3323 633 619 745 232 993 Family Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Peridiniaceae Pfiesteriaceae Pfiesteriaceae Pfiesteriaceae Prorocentraceae Prorocentraceae Prorocentraceae Prorocentraceae Prorocentraceae Symbiodiniaceae Symbiodiniaceae Order Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Peridiniales Prorocentrales Prorocentrales Prorocentrales Prorocentrales Prorocentrales Suessiales Suessiales Haemosporida Table 7b: List of dinoflagellate species a nd GenBank accession number for ITS analysis Dinoflagellate species Pentapharsodinium dalei SZN19 Pentapharsodinium tyrrhenicum Peridinium aciculiferum strain PASP6 Peridinium cinctum SAG: 2017 Peridinium willei Pfiesteria piscicida clone CCMP 2091 Pfiesteria shumwayae clone CCMP 2359 Pfiesteria-like sp CCMP 1838 Prorocentrum belizeanum Prorocentrum cassubicum strain VGO 835 Prorocentrum levis Prorocentrum micans strain PM1V Prorocentrum minimum strain AND3V Sybiodinium sp. ex Amphisorus hemprichii Symbiodinium sp. clade A Plasmodium vivax (outgroup) 58 Gen Bank No. EF036546.1 47.1 30.1 26.1 33.1 32.1 340.1 EF506569.1 63.1 19.2 93.1 17.1 38.1 EF036562.1 74.1 85.1 87.1 EU130568.1 EF0365 AB2901 AB2901 EU1261 EU1261 (this study) EF417 EF0365 AF3575 AF5025 AY4561 AY7452 DQ336067.1 EU1305 DQ0829 AJ2987 BPs 925 932 9 9 9 9 9 82 847 9 11 9 9 9 932 9 9 32 40 40 48 36 751 8 26 12 26 26 43 949 38 38 1131 Family Ceratiaceae ac c c c c e Peridiniaceae e e e e c Pyrocystaceae niac iac Dinophysiaceae Ceratocory eae Gonyaulaca eae Gonyaulaca eae Heterocapsa eae Heterocapsa eae Peridiniaceae Peridiniac ae Peridiniac ae Pfiesteriac ae Pfiesteriac ae Pfiesteriac ae Prorocentra eae Prorocentraceae Symbiodi eae Symbiodin eae Order Gonyaulacales c c c Peridiniales ia ia ia ia tr Pyrocystales ales ales pori Dinophysiales Gonyaula ales Gonyaula ales Gonyaula ales Peridinia les Peridinia les Peridiniales Peridinia les Peridin les Peridin les Peridin les Peridin les Prorocen ales Prorocentrales Suessi Suessi Haemos da Table 8: List of dinoflagellate species and GenBank accession number for Cty b analysis Dinoflagellate species Ceratium longipes A yaulax se Hetero CCMP 15 Hete CMP 44 Scrippsiella aff. hangoei K-0399 yae a e 827 Proro CMP 1589 Pl (outgroup) Dinophysis acuminata Ceratocorys horrida lexandrium pseudogon Alexandrium tamaren capsa rotundata strain 42 rocapsa triquetra strain C 9 Pentapharsodinium sp. Peridinium centenniale CCAC0002 Scrippsiella sweene Pfiesteria piscicid Pfiesteria shumwaya Pfiesteria-like sp CCMP 1 centrum micans strain C Prorocentrum minimum strain JA0001 Pyrocystis noctiluca Symbiodinium goreaui Symbiodinium microadriaticum asmodium falciparum 59 Results rDN18S n sA a aly is nd, One th a f h r i n b . u e e b e o r v tly from the host, as well as from cultured ls q c b ither 1 n l e u e i nBank e m r o d r fact, ence comparisons with P. tyrrhenicum revealed i d r e n . c t d o u n n biguity code of K for arasitic dinoflagellate. The second difference was located at nucleotide 1062 of P. enicu, resulting in an ambiguity code of W and a code of T fo e asitic flag e s otide resulted in a code of C and an f o e e a e s a s species a h ( w e n ithin r ( . Here, the bootstrap values in the 18S rDNA provided strong support for the rity of the families presented including the Gonyaulacacea, Heterocapsaceae, fiesteriaceae, Prorocentraceae, and Symbiodiniaceae. Also the 18S rDNA analysis gives rong bootstrap values (i.e. >85%) to several unclassified dinoflagellates, Azadinium poporum and Azadinium spinosum, supporting their claim as a monophyletic group. The analysis also confirms the placement of Duboscquodinium collinii with the Scrippsiella ous ive und ed s xty ine ase pairs of the SSU rDNA were isolated from direc sour nr/n sequ of w resu the p tyrrh dino amb is co phyl majo the parasitic dinoflagellate Seq enc s w re o tain d fr m both cells emo ed cel pari taph . Se son b arso a difference of only three bases, all uen y BL iniu es o AST m ty tain n w rhen ed fr th th icum om e e Ge ; in ce w t dat ere abas 00% retu ide rned tica a 99 . Th % si seq ila enc ity t com Pen hich lted wer in an e am amb bigu igui ties. y co The e of first W f iffe r P. enc tyrrh , at enic ucle m a otide d a 212 am of P tyrrheni um, r th s of t al. par the 2009 ellat ty co imil only etic . La de o rity acce affil tly, M f in 18 pted iatio nucle r th S rD to be is w 134 asitic sequ ter t the 4 of dino nce an 9 Orde P. ty flag obt 5% r Pe rrhen llate ined Caro idini icum . from n, C ales igui S mm ogen par NA gre org ount Fig anism ay 26). ame ). The P st 60 18S rDNA CGGCAAAACTGCGAATGGCTCATTAAAACAGTTATAGTTTATTTGATGGT CATTCTTTACATGGATAACCGTGGTAATTCTAGAGCTAATACATGCGCCC AAACCCGACTCCGTGGAAGGGTTGTGTTTATTAGKTACAGAACCAACCCA GGCTCTGCCTGGTCTTGTGGTGATTCATAATAACCAAACGAATCGCATGG CATCAGCTGGCGATGAATCATTCAAGTTTCTGACCTATCAGCTTCCGACG GTAGGGTATTGGCCTACCGTGGCAATGACGGGTAACGGAGAATTAGGGTT CGATT A A GCCGGAGAGGGAGCCTG GA AC GCTACCACATCTAAGGAAGGCA GCAGGCGCGCAAATTACCCAATCCTGACACAGGGAGGTAGTGACAAGAAA TAACAATACAGGGCATCCATGTCTTGTAATTGGAATGAGTAGAATTTAAA TCCCTTTACGAGTATCGATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGG TAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCGGTTAAAAAGCTC GTAGTTGGATTTCTGCTGAGGACGACCGGTCCGCCCTCT GTATC GGGTGA TGGCTCGGCCTGGGCATCTTCTTGGAGAACGTAGCTGCACTTGACTGTGT GGTGCGGTATCCAAGACTTTTACTTTGAGGAAATTAGAGTGTTTCAAGCA GGCACACGCCTTGAATACATTAGCATGGAATAATAAGATAGGACCTCGGT TCTATTTTGTTGGTTTCT A AG GCTGAGGTAATGATTAATAGGGATAGTTG GGGGCATTCGTATTTAACTGTCAGAGGTGAAATTCTTGGATTTGTTAAAG ACGGACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTGATCAAGAAC GAAAGTTAGGGGATCGAAGACGATCAGATACCGTCCTAGTCTTAACCATA AACCATGC TCT CAGCGACTAGAGATTGGAGGTCGTTA TTACGACTCCTT C ACCTTATGAGAAATCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTCGCAA GGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCT GCGGCTTAATTTGACTCAACACGGGGAAACTTACCAGGTCCAGACATAGT AAGGATTGACAGATTGATAGCTCTTTCTTGATTCTATGGGTGGTGGTGCA TGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTAATTCCGTTAACGA ACGAGACCTTAACCTGMTAAATAGTTACACGTAACCTCGGTTACGTGGGC AACTTCTT GGG CG CGCAAGGA CAAT AGA ACTTTG TGTCTAA AGTTTGAGG AACAGGTCTGTGATGCCCTTAGATGTTCTGGGCTGCACGCGCGCTACACT GATGCGCTCAACGAGTTTATGACCTTGCCCGGAAGGGTTGGGTAATCTTT TTAAAACGCATCGTGATGGGGATAGATTATTGCAATTATTAATCTTCAAC GAGGAATTCCTAGTAAGCGCGAGTCATCAGCTCGTGCTGATTACGTCCCT GCCCTTTGTACACACCGCC Table 9: Consensus sequence for 18S rDNA gene sequences from the parasitic inoflagellate isolate. d 61 group, inferred by Coats?s recent redistribution of the dinoflagellate out of the he Dinophyseae (Coats, Kim et al. 2010). The Family Peridiniaceae, which i Syndiniophyceae to t ncludes Pentapharsodinium, Ensiculifera, and Scrippsiella is grouped relative to genus. The Pfiesteria group, Family Pfiesteriaceae, divides the Peridiniaceae, but is grouped as a single Family. Thus, Pentapharsodinium and Ensiculifera form a monophyletic group. Figure 26. SSU rDNA Maximum likelihood 1,000 bootstrap replicates using the GTR model. The tree is a representation of phylogenetic relationships across various Orders of closely related dinoflagellates. 62 Internal transcribed spacer region analysis. Fi he rasitic d from st and S ve hundred ninety three base pairs of ITS sequence were obtained from t pa dinoflagellate. Sequences were obtained from both cells that were remove ho cells in culture. Sequences obtained from either source were identical. IT TCATTCGCACGCATCCAAATGAACCACTGTGAATCATTGGCGTGAGGTTC TGCATGGGGGACGGAGATTGCATCAATTCCCCCATGCAGAAGCTCGAGGG CGGCAGGGCAGGATGGGTGTTTGTCACCTCCTTTCTGTTCTTGTCGTCAT GTACCTTGCATGCTGATCTTTACATCCTCATGAACTCTGGAGTGCTTGCC CACTCCTTTTTCTTTCTTACAACTTTCAGCGACGGATGTCTCGGCTCGAA CAACGATGAAGGGCGCAGCGAAGTGTGATAAGCATTGTGAATTGCAGAAT TCCGTGAACCAATAGGGACTTGAACGTACACTGCGCTTTCGGGATATCCC TGAAAGCATGCCTGCTTCAGTGTCTATTCCATCTTCTGCCAGTGACGTCT TCCACCTCGTGTGGTCCAGTCGCTTGTGCGTGCTTGTGCGTTAAGGAGCT GTGCTGCCCCTGACGCATTCAGTGCATGGGGAGTTTCCGTGACTTGCAAC TTACCATACATTGCTGATGTTATTTGTTGCTGTGCCACTGGAAAGAGCCC TTGTGTGGAGTATGTCTCATACTTCTCTAAGACATGAAGTTAG Table 10 ate olate. In of exico, N uence mparis , and ne trans t for l of the eterocap e only NA nalysis the two genera Pentapharsodinium and Ensiculifera both belong to the Family Peridiniaceae, and form a monophyletic group. : Consensus sequence for ITS sequences from the parasitic dinoflagell is contrast, the sequences from specimens obtained from the Northern Gulf M orth Carolina and the Gulf of Naples, Italy were only 94% identical. Seq co ons with P. tyrrhenicum showed a total of three gaps, eighteen transitions ni versions. The bootstrap values in the ITS analysis provided strong suppor al major Families presented including the Dinophysiaceae, Gonyaulacacea, H saceae, Pfiesteriaceae, Prorocentraceae, Symbiodiniaceae and Peridiniaceae. Th notable exception is with the genus Peridinium. Its placement with Pfiesteriaceae and Prorocentraceae is likely to be artifactual. As seen in the 18S rD a 63 Figure 27 ITS Maximum likelihood search with 1,000 replicates using GTR model. The tree is a representation of phylogenetic relationships across various Orders of closely related dinoflagellates. 64 Molecular analysis cytochrome b (cyt b). Seven hundred fifty one base pairs of cyt b sequence were obtained from the parasitic dinoflagellate. Sequences were obtained from both cells that were removed from host and cells in culture. The sequences obtained from both host and cultures were yt b TGGAATTACTATTATATTACAAATTATTACTGGAATCTTATTATCTTTAC C ATTATACTTCAGATATTAATAGTGCTTACTTCTCTATATTCTTTATTATA AGAGAAATATTCTTTGGATGGTCTTTACGTTATTTACATTCTTCAGGTGC ATCATTTGTATTCTTATTTGTATTCTTTCATATTGGAAGAGGTATATTTT ATGGTTCATATTTCTATAATCCAAATACTTGGTTTTCTGGTATTATTCTT TTATTATTTTTAATGGCTATAGCATTTATGGGTTATGTCTTACCTTTTGG ACAAATGAGTTTCTGGGGAGCTACAGTAATTACAAATTTATTATCACCTT TTCCATGTGTAATAGAATGGGTTTCTGGAGGATATTATGTTTACAATCCA ACTTTAAAGAGATTTTTTATATTCCATTTCTTATTACCATTTCTATTATG TGGATTTACTATTCTTCATATTTTTTATCTTCATTTACTATCTTCTAATA ATCCTTTAAGGAATTCTACTAATAATAAAATCCCATTTTTCCCTTATATA TTTCAAAAAGATGTATTTGGTTTCATTATAATCCTTACTATATATCTTCT TCAAACTAATTTTGGTATATCTTCTTTATCACATCCAGATAATGCATTAG AAGTTTGTTCCTTACTTACTCCTTTACATATAGTACCTGAATGGTATTTC CTATGCCAATATGCTATGTTAAAAGCTGTACCCAACAAAAATTCAGGATT C Table 11: Consensus sequence for cyt b gene sequences from the parasitic is arasitic dinoflagellate isolate. identical. The sequence comparison by BLASTn against the GenBank nr/nt database returned relatively low homology with several dinoflagellate genus belonging to the Order Peridiniales in the 86% - 88% range. The bootstrap values in the cyt b analys provided support for the Families Gonyaulacaceae, Heterocapsaceae, Pfiesteriaceae, Prorocentraceae, Peridiniaceae, and Symbiodiniaceae. The phylogeny of the p dinoflagellate is not easily resolved due to the lack of taxa available for analysis. However, the parasitic dinoflagellate does fall within Order Peridiniales. 65 66 Figure 28 Cyt b analysis based on maximum likelihood, with 1,000 replicates using GTR model. The tree is a representation of phylogenetic relationships across various Orders of closely related dinoflagellates. Discussion and Conclusions It is instructive to recall that analyses of the cell morphology indicates that the parasitic dinoflagellate could be described as a Pentapharsodinium species and it is morphologically indistinguishable from P. tyrrhenicum, except for the antapical plat es, wo ont. The olecular analyses appear to weaken that conclusion. Th tic group ig 26 an logenetic alysis u genera ntapha Fig. 29) ottschli gical alyses o trong idence rso- nium an nsiculifera (Fig. 30) (D'Onofrio, Marino et al. 1999). S rDNA places the parasitic dinoflagellate within the E/Pe clade. ies y s. ould clade. It may be ndergoing rapid evolution. In the study mentioned earlier by the Gottschling group which uld be the ones most likely to be modified for attachment by the troph m e two genera Pentapharsodinium and Ensiculifera sit in a monophyle (f d 27). In another study the Gottschling group concluded, based on phy an sing ITS, 5.8S rRNA, and domains D1 and D2 of the LSU, that the two Pe rsodinium and Ensiculifera form a monophyletic clade, termed E/Pe ( (G ng, Renner et al. 2008). The results of a study using ITS and morpholo an f multiple calcareous dinoflagellates were also interpreted to provide s ev of monophyly and even showed the mixing of genera among Pentapha di d E In this study, 18 The parasitic dinoflagellate falls within that clade with other Pentapharsodinium spec and so is tentatively recognized as a Pentapharsodinium species. The strong homolog between P. tyrrhenicum and the parasitic dinoflagellate, based on 18S rDNA, could suggest that the parasitic dinoflagellate is a variant of P. tyrrhenicum. Other Pentapharsodinium species have not been officially reported in North American water The observation that several Ensiculifera have been found in the Gulf of Mexico c suggest that the parasitic dinoflagellate is a species belonging to the E/Pe u 67 (Gottschling, Renner et al. 2008) evidence is presented suggesting that Pentapharsodinium tyrrhenicum evolved from a lineage that included the Ensiculifera genus sometime during the Cretaceous (Fig. 31). ITS analyses showed the parasitic dinoflagellate appears to fit within the E/Pe clade (Fig. 27). ITS is used in phylogenetics to resolve genetic drift between populations. In this case, it reveals the mixing of genera on the E/Pe clade. Internal transcribed spacers 1 and 2 are introns known for particularly high rates of mutation (Blouin 2002). The variability within the ITS regions arises from the lack of evolutionary constraint. This occurs because they do not encode for a protein that can be acted upon by natural selection, and therefore can change without consequence for fitness. The mixing of genera could suggest that this molecularly and morphologically closely related group may need to be recharacterized and that members belonging to either taxa may be incorrectly categorized. Molecular analysis based on ITS in other studies in the E/Pe clade (D'Onofrio, Marino et al. 1999) provide evidence of genera mixing, and suggest that E. imariensis could be a species of Pentapharsodinium (Fig. 30). In another study involving ITS analysis (Hai-Feng and Yan 2007) the investigators placed a previously undescribed dinoflagellate into Ensiculifera, although the cell possessed the morphological plate tabulation of Pentapharsodinium. Due to inconsistencies within the aceae. Cytochrome b is a mitochondrial gene used as part of the electron transport chain involved in cellular respiration and the production of ATP. The mutation rates of mitochondrial genes are known to be more elevated than many nuclear genes (Brown, E/Pe clade I feel that the resolvability of the ITS analysis can only show accurately that the dinoflagellate is a member of the Family Peridini 68 George et al. 1979). The cause of a higher mutation rate is though to be attributed to the replication and the lack of enzymatic capability to remove or repa lem arose from the lack of taxa needed to do a thorough phylogenetic analysis based on the cyt b gene. My analyses were editing function of the mtDNA ir thymine dimers (Lansman and Clayton 1975). Therefore, changes in mitochondrial genes give a fine-tuned molecular clock, allowing for taxonomic assignment even beyond the species level. In this study, analyses of cytochrome b gene sequences were unable to resolve the parasitic dinoflagellate to the level of Family. The prob able to resolve the parasitic dinoflagellate only to the level of Order Peridiniales (Fig. 28). 69 Figure 29. Maximum Likelihood (ML) phylogeny for calcareous dinoflagellates helices I and II of ITS1. Numbers above branches indicate ML bootstrap based on sequences of the LSU rRNA domains D1and D2, the 5.8S rRNA, and support from 1,000 replicates GTR model (Gottschling, Renner et al. 2008). 70 Figure 30 The phylogenetic trees obtained using ITS1 and ITS2 sequences. Numbers at internal branches indicate percentage of bootstrap (500 replicates); values < 50% have not been included. (a) Neighbor-joining analysis; scale bar = 10% divergence. (b) Parsimony analysis; scale bar = 10 steps (D'Onofrio, Marino et al. 1999). Figure 31. Chronogram depicting the separation of the E/Pe clade into separate genera during the Cretaceous period (D'Onofrio, Marino et al. 1999). 71 Overall conclusions The molecular analyses presented here provide strong multiple levels of support that the parasitic dinoflagellate is a member of Order Peridiniales, Family Peridinaceae, and could provisionally be identified as a member of genus Pentapharsodinium. The molecular analyses, particularly at the 18S level, suggest near-identity with P. tyrrhenicum, but there is insufficient resolving power with the ITS and cyt b analyses to provide finer resolution than at the Family level. Morphological analyses strongly suggest very close affinities to P. tyrrhenicum; yet the antapical plates strongly suggest that this cell is a distinctly different organism from the benthic autotroph described in 1993 by Montressor et al. It is instructive to consider that the antapical plates, which are the only ones that show morphological differences, would of course be the plates that would be most likely to be modified in an organism that exhibits cell attachment and nutrient capture via a ventroposterially emergent peduncle. Analyses of the behavior and life cycle indicate that this organism is a mixotrophic dinoflagellate capable of parasitizing a ctenophore in a very specific tenoph re is prey to several species of fish (including Menidia beryllina, Petrilus burti, nd Mugil cephalus) or the common sea nettle (Chrysaora quinquecirrha), a schyphozoan which is a major predator on Mnemiopsis (Moss, pers. communication). In addition, it has never been seen on other ctenphores The dinoflagellate is not an obligate parasite as evidenced by my ability to culture it in vitro. host/parasite relationship. To date, since its first discovery in the early 1990s by Moss and colleagues, it has not been observed on any other host, despite the fact that the host c o a 72 Acknowledgements The research described herein was aided greatly by my lab mates Matthew Dodson and Christopher Taylor, James Gillespie and the numerous undergraduates who have helped in so many different ways. The work could not have proceeded without the intellectual and materiel support of Dr. Scott Santos and Dr. Mark Liles, and to my advisor for providing the facilities used throughout this project. I thank the staff of the NERRS palachicola Site, especially Dr. Edmiston, Jenna Wanat and Lauren Levi. Funding for Environmental Signal Transduction (CECST) program of the College of Science and Mathematics, R.P. Henry and F.F. Bartol. We thank Dr. Barbara Sullivan and Debbie VanKeuren for help in the early stages. Some travel money was made available by the College of Sciences and Mathematics Graduate Travel Awards Committee. Dr. Eric Wommack, Dr. Wayne Coats and Dr. Feng Chen graciously allowed me to join the MOVE06, MOVE07 and MOVE08 cruises on board the R/V Cape Henlopen and R/V Hugh Sharp; I am also grateful to the students of that cruise for their comradeship during difficult conditions; their cheery demeanors kept the work moving forward. I am also grateful to Dr. Debra Bronk for housing at VIMS during the first of those cruises. I am forever grateful to my loving wife Amy and my kids, Malana, Makayla, and Maliki. A the project was provided by a grant from the National Science Foundation MCB-0348327 and two Research Grant in Aids from the College of Science and Mathematics to my advisor, Dr. Anthony Moss, and NSF-EPSCoR NSF EPS 0447675 to the Cellular and 73 Appendix: 1 Molecular Analysis of Trichodina ctenophorii and an unknown amoeba associated to the comb plates of Mnemiopsis ns ic primers C-3? that amplify the flanking regions of the 1 or the oratory of the Auburn Research Instrumentation Facility on an ABI 33100 produce rbor, During the course of this study some molecular and ultrastructural (SEM) work was performed on the other protists found associated with Mnemiopsis. DNA extractio and PCR amplifications were as described in chapter 4. The universal eukaryot 18ScomF1 (forward) 5?-GCTTGTCTCAAAGATTAAGCCATGC-3? and 18ScomR1 (reverse) 5?-CACCTACGGAAACCTTGTTACGA 8S rDNA gene (Zhang, Bhattacharya et al. 2005) were used to amplify the 18S rDNA region of the organisms. Trichodina ctenophorii were removed from the host by either direct pipetting treating with a 100mM KCl solution. Cells were washed several times in sterilized artificial seawater to remove any ctenophore tissue and collected into acetone. After DNA extraction, PCR was performed, producing amplicons that were sequenced at Genetic Analysis Lab sequencer. The chromatograms were visualized, edited and assembled to consensus sequences using Sequencher (Gene Codes Corporation Ver. 4.8, Ann A MI, USA). Contiguous sequences and sequences obtained from GenBank were aligned using ClustalX ver. 2.0.10 (Larkin, Blackshields et al. 2007) set on Multiple Alignment Mode. ClustalX was then used to construct a neighbor-joining phylogenetic tree. 74 18SCOMF1 TGGGCTTAATCTTTGAGACAAGCAGTTGCGTGGACTCATAGTAACTGATCG GATCGCTTCGGCGATGAGTCATTCAAGTTTCTGCCCTATCAGCTTTGATGG TAGTGTATTGGACTACCATGGCAGTCACGGGTAACGGAGAATTAGGGTTCG GTTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCTAAGGAAGGCAGCA GGCGCGTAAATTACCCAATCCTGATTCAGGGAGGTAGTGACAAGAAATAAC AACCTGGGGCTTTGCTTTCGGGATTGCAATGATCGTAATCTAAAGCAATTA GAAAGAAACCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCA GCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTCAA CTTCTGCCCCGGGGCCGAGAGGCGACTCGGAGGTCCCGGGGCATCCGTTCC GCACCACGTCTACGCGTGAGGGCGGACAGTTTACCTTGAGAAAATTAGAGT GTTCACGCAGGCGTAGCCAGTATACATTAGCATGGTATATGGTAAGAGGAC TCCAAGCCGTTGTTGGT Table 12: Contiguous sequence of Trichodina ctenophorii using primer 18SCOMF1 18SCOMR1 GGGACGTAATCAGCGCAAGCTGATGACTTGCGCTTACTAGGAATTCCTCGT TCACGACCCATAATTGCAAGGGTCGATCCCAATCACGGCACACCCTGACAG GTTACCCGGCTCCCTTCGGATCAGGAAACTCGCTGTGTGTGCCATTGTAGC GCGCGTGCGGCCCAGGACATCTAAGGGCATCACAGACCTGTTATTGCCTCA AACTTCCGTGCGATAGGCTCGCACAGTCCCTCTAAGAAGCACCTTCCGTTG AGACGGGGTGCTAGTTAGCAGGTTAAGGTCTCGTTCGTTAAAGGAATTAAC CTGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCGTAGA ATCAAGAAAGAGCTCTCAATCTGTCCATCACACCCACGTTTTGACCTGGTA AGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTG CCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAG AACCCAAAGACTTTGATTTCTCGTACGGACCCAGCCAGGGACAATCCCTGA CTGAATCCGAGTCGGTATGGTTTATGGTTTAGGACTAGGACGGTATCTGAT CGTCTGTGATCCCCTAACTTTCGTTCTTGATCAATGAAAACATCCTT Table 13: Contiguous sequences of Trichodina ctenophorii using primer 18SCOM Trichodina species R1 BPs Gen Bank No. Trichodina ctenophorii 1237 (this study) Trichodina sinipercae 1704 EF599288.1 Trichodina hypsilepis 1693 EF524274.1 Trichodina heterodentata 1698 AY788099.1 Trichodina reticulate 1702 AY741784.1 Trichodina sp. LAH-2003a 764 AY363960.1 Trichodina nobilis 1698 AY102172.1 Cryptomonas paramecium 1984 AJ420676.2 Table 14: List of Trichodina species and GenBank accession numbers 75 Trichodina heterodentata Trichodina nobilis Trichodina sp. LAH-2003a Trichodina reticulata Trichodina ctenophorii Trichodina sinipercae Trichodina hypsilepis Cryptomonas paramaecium ina nder the family Trichodinidae. The phylogeny of T. ctenophorii may not be completely ccurate due to the lack of a complete gene sequence. However, based on the presented ata it is clear that T. ctenophorii belongs to the genus Trichodina. The Flabellula ? like gymnamoebae associated with the comb plates of Mnemiopsis may possibly be an unknown marine stramenopile. The universal eukaryotic Figure 32 Neighbor Joining Tree of Trichodina showing that Trichodina Ctenophorii sits within the Trichodina group. The resulting data placed Trichodina ctenophorii within the genus Trichod u a d 76 primers mentioned previously were utilized in the molecular analysis (Table 13). Amoebae w Cells were e used for DN Primer ere collected from cultures grown in MY100 medium at room temperature. centrifuged at 10,000g and the medium decanted off. The pelleted cells wer A extraction as described in chapter 4. Sequence Region Specificity Reference 23FPL publication et 5?- (Barns, GCGGATCCGCGGCCGCTGCA 18S rDNA Not specified in Fundyga GAYCTGGTY GATYCTGCC-3? al. 1994) 518R 5 (Muyzer, de '-ATTACCGCGGCTGCTGG-3' 18S rDNA V3 region of Waal et al. 16S rDNA 1993) SimF A . 5'- encompass the (Sims, l YCTGGTTGATYYTGCCAG-3' 18S rDNA V1, V2, V3, Aitken et a Universal primers that V4, V7, and V8 2002) regions SimR T l. 5'- encompass the (Sims, GATCCATCTGCAGGTTCACC 18S rDNA V1, V2, V3, Aitken et a 2002) T-3' V4, V7, and V8 Universal primers that regions 18ScomF 5?- GCTTGTCTCAAAGATTAAGC -3? 18S rDNA Flanking regions of the NA (Zhang, Bhattacharya et al. 2005)CATGC 18S rD 18ScomR CCTTGTT S rD king of the DNA (Zhang, Bhattacharya et al. 2005) CACCTACGGAAA 5?- A 18 CGAC-3 NA regions Flan 18S r Table 15: List of PCR primers for Flabe like e analysis llula ? gymnamoeba 77 78 18ScomF1 TGTCAGTTAAGCGACTTTTTACTGTGAACTGTGAACGGSTCATTAC ATCG GTTCTAGTCTCTTTGGTAGTTCATCGTGTGTGTCATCTTCCCTTTCG GGG AGAGCACGCAAGGTTTARTTGGATAACTGTCATAATTTGAGAGCT AATAC ATGCCTAAAAGTCCTCGGTTGCTGCTTTTTGCAGGGATGGGGATGC GTTT ATTARATTGAGACCGGAGGCGCGCAAGCGTCGTTTTGTAAGGTGA CTCAC AATAACCACTCGGATCGCTCTTCGTGAGCGATGTACCATTCSAGTT TCCG TCCTATCATGCTTGGAAGGKAAGGTATCGGCTTACCTTGGCGTTAA CGGG CAACGGARAATTAGGGTTCGGTTCCGGARAGGGGGCCTGAGACAT GGCCA CCACATCCAAGGAAGGCAGCAGGCGCGTAAATTACCCAATCCTAA CTCAG GGAGGTAGTGACAATAACTAACGATGGTGCGCGCATGTTCCGTTT ATCGG AAGATCGTACACCAATCGTCATGAGAACAATCTAAACACCTTATC GAGGA ACCATTGGAGGGCCAGTCTGGTGCCAG Table 16: Contiguous sequences of Flabellula ? like gymnamoebae using primer 18SCOMF1. 79 23FPL GACT TCATACGCTTGTCTCAAGATTAAAGCCATGCATGTCAAGTTAAAGC TTTTAACTGTGAAACTGTGAACGGCTCATTACATCGGTTCTAGTCTC TTT GGGAGTTCATCGTGTGTGTCATCTTCCCTTTCGGGGAGAGCACGCAA GGT TTAGTTGGA TCATAATTT CTA CC GTCC TAACTG GAGAG ATACATG TAAAA T A ACC CGGTTGCTGCTTTTTGCAGGG TGGGGATGCGTTTATTAGATTGAG GGAGGCGCGCAAGCGTCGTTTTGTAAGGAGACTCACAATAACCACT CGGA T C TT TA TTG CGCTCTTCGTGAGCGATGTAC ATTCGAG TCCGTCC TCATGC GAAGGTAAGGTATCGGCTTACCTTGGCGTTAACGGGCAACGGAGAA TTAG G A G GTTCGGTTCCGGAGAGGGGGC GAA CTGAGAC TGGCCACCACATCCAA GGCAGCAGGCGCGTAAATTACCCAATCCTAACTCAGGGAGGTAGTG ACAA TAACTAACGATGGTGCGCGCATGTTCCGTTTATCGGAAGATCGTAC ACCA A TTCGTCATGAGAACAATCTAAACACCTTA CGAGGAACCATTGGA Table 17: Contiguous sequences of Flabellula ? like gym pr 23FPL. namoebae using imer 80 T 518R A GGCCTCCATGGTTCCTCGATAAGGTGTTTAGATTGTTCTCATGACG TT G T GTGTACGATCTTCCGATAAACGGAACATGCGCGCACCATCGTTAG TAT TGTCACTACCTCCCTGAGTTAGGATTGGGTAATTTACGCGCCTGCTG CCT T C CCTTGGATGTGGTGGCCATGTCTCAGGCCCCCTCTCCGGAACCGAA CC TAATTCTCCGTTGCCCGTTAACGCCAAGGTAAGCCGATACCTTACCT TCC A GCGAT AGCATGATAGGACGGAAACTCGAATGGTACATCGCTCACGAAGA C C CGAGTGGTTATTGTGAGTCACCTTACAAAACGACGCTTGCGCGCCT CG G C TCTCAATCTAATAAACGCATCCCCATCCCTGCAAAAAGCAGCAAC GAG G A ACTTYTAGGCATGTATTAGCTCTCAAATTATGACAGTTATCCAACT AA CCTTGCGTGCTCTCCCCGAAAGGGAAGATGACACACACGATGAACT ACCA A A AGAGACTAGAACCGATGTAATGAGCCGTTCACAGTTTCACAGTTA AAA TGA GTCGCTTTAACTTGACATGCATGGCTTTAATCTTTGAGACAAGCGTA Table 18: Contiguous sequences of Flabellula ? like gymnamoebae using primer 18R. 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