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THE COELOMOCYTES AND INFLAMMATION IN THE SEA URCHIN LYTECHINUS VAR I EGATUS (LAMARCK, 1816) BY Katrina Bang A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Professor Sandra Gilchrist Sarasota, Florida January 2012
i ACKNOWLEDGEMENTS When I began this thesis I did not understand how much work I was getting myself into : it was after spending many long nights in lab and after slaving away writing this thesis that I realize d the complexity of the project. In the end I learned that I am capable of working incredibly hard on a difficult project, and it showed me th at I can complete a n ambitious project on a tight schedule There are a few people whom I seriously thank for all of their help and encouragement throughout th is project : without you all, I would not have been able to get Hannah Schotman cold November o cean for spending long days and nights helping me experiment in lab, for helping me proofread/format my thesis, and most of all thank you for your friendship y ou helped me survive my thesis year Mom You are my rock and a voice of reason in my life. Thank you for listening to me in the mid st of many thesis meltdowns and for visiting me and making all that bomb diggity vegetarian food when I was so stressed Dad Thanks for trekking up to Sarasota just before my baccalaureate exam and pulling all nighters with me. Also, thanks for advising me to ditch my work for a few ho urs to go eat shellfish and drink beer with you that one night: I needed it more than I kne w. My thesis committee A big thanks for your flex ibility, guidance, and support And finally M y thesis Thank you for teaching me that I am strong and capable; even though I hated you in the end.
ii THE COELOMOCYTES AND INFLAMMATION IN THE SEA URCHIN LYTECHINUS VAREIGATUS (LAMARCK, 1816) Katrina Bang New College of Florida, 2012 ABSTRACT Phagocytosis is a first line of host defense i n vertebrate and invertebrate immunity. A report that body wall from the earthworm Lumbricus terrestris contained an inhibitor of L. terrestris phagocyte migration, prompted the question of whether tissues from the sea urchin Lytechinus variegatus similarl y affected the migration, and consequently, the ability of L. variegatus phagocytes to eliminate foreign invaders Phagocytotic coelomocytes from L. variegatus were studied in vitro using light microscopy to gain an understanding of the ions and morphologies. Tissues were harvested from the digestive tract, gonads, peristomial gills, and peristomial membranes of L. variegatus urchins, and each tissue individually assayed to determine their effect on the phagocytotic coelomocytes. The effe ct of L. variegatus tissues on
iii phagocytes and inflammation was evaluated by analyzing changes in the median number of total phagocytes (TP) and the phagocytosis index (PI) of phagocytes over time. Preliminary observations of the cells in vitro indicate tha t small phagocytes may initiate and augment the formation of cellular clots within the coelomic fluid via a net like mechanism. In the presence of yeast in vitro a significant number of phagocytes were found to migrate into peristomial gills Statistic al analysis indicated that the presence of yeast may induce an increase in the TP in viv o; significant differences were not found between the PIs from any of the experimental conditions analyzed. These findings suggest that the presence of yeast causes inf lammation, and that phagocytes migrate into peristomial g ills following phagocytosis and encapsulation. _____________________________________________________________________ ______________________ Dr. Sandra Gilchrist Division of Natural Sciences
iv TABLE OF CONTENTS Acknowledgements i Abstract i i Chapter 1 Introduction 1 I. An Introduction to the Coelomocyte 5 II. Vibratile Cells 9 III. Amoebocytes 11 IV. Phagocytes 1 5 A. What is the phagocyte? 1 5 B. Petaloid vs. Filopodial 1 6 C. Clotting 1 8 D. Ch emotaxis 20 E. Phagocytosis 22 F. Lytechinus variegatus 2 4 Chapter 2 Methods 32 I. General 3 2 A. Urchin Collection and Maintenance 32 B. Removal of Coelomic Fluid 33 C. Preparation of Heat Sterilized Yeast 34 i. Yeast Staining with Eosin Y 34 ii. Preparation of Eo sin Y yeast to 1,8000 yeast/mL FSW 35 D. Yeast Counting 3 5 E. Determination of Coelomocyte Density 3 6 II. Experimental 3 7 A. Observations of Coelomocytes In Vitro 3 7 i. Observations of Coelomocytes in Wet Mounts 3 7 ii. Observations of Coelomocytes in Hanging Drops 3 7 B. P hagocytosis Assays 3 8 i. Experiment 1: In Vivo Phagocytosis Assays 3 8 ii. In V itro Phagocytosis Assays 41 a. Experiment 2: Assays Without Urchin Tissue 41 b. Experiment 3: Assays With Urchin Tissue 44 C. Analysis of Phagocyte Activity from Stained Smears 4 8 i. A Coord inate Grid for Cell Counts 4 8 D. Equations 4 9 Chapter 3 Results 50
v I. Observations of the Coelomocytes 50 II. Phagocytosis Assays 5 4 A. Statistics 5 5 B. Phagocytosis Assays 56 Chapter 4 Discussion 5 8 I. Observations of the Coelomocytes 5 8 II. Phagocy tosis Assays 59 III. Directions for Future Studies 6 2 IV. Conclusions 6 4 Cited References 6 6 Appendix I Solutions Recipes 7 3 Appendix 2 Instructions for Urchin Tissue Harvest and Preparation 7 4
vi LIST OF FIGURES Figure 1: The Phylogeny of Extant Phyla and Classes 2 Figure 2: The Urchin Anatomy 3 Figure 3: Comparison of Mammalian and Echinoid Immune Cells 4 Figure 4: Comparison of P hagocytotic Coelomocyte to a Human Macrophage 5 Figure 5 : Image of a Vibratile Cell 1 0 Figure 6: Images of Amoebocytes 12 Figure 7: Immune Functions of Phagocytes 15 Figure 8 : Petaloid and Filopodial Phagocytes In Vitro 1 7 Figure 9 : Urchin Anatomy vs. L. terrestris Anatomy 1 9 Figure 10: Urchin Collection Site 3 3 Figure 11 : Diagram Illustrating Yeast Counts 3 6 Figure 12 : Flow Chart for Experiment 1: In Vivo Phagocytosis Assay 40 Figure 13 : Flow Chart for Experiment 2: In Vitro Phagocytosis Assay (Steps 1 4) 43 Figure 1 4 : Flow Chart for Experiment 2: In Vitro Phagocytosis Assay (Steps 5 10 ) 44 Figure 1 5 : Flow Chart for Experiment 3: In Vitro Phagocytosis Ass ay w ith Tissues (Steps 1 5) 4 6 Figure 1 6 : Flow Chart for Experiment 3: In Vitro Phagocytosis Ass ay w ith Tissues (Steps 6 14 ) 47 Figure 1 7 : Diagram of Grid Construct Used for Cell Counts 4 8 Figure 1 8 : Images of Coelomocytes from Observations 5 1 Figure 19 : Images of A Red Amoebocyte from Observations 53
vii Figure 2 0 : Diagram for Tissue Harvest from L. variegatus 7 5
viii LIST OF TABLES Table 1: Summary of Coelomocyte Morphologies and Functions 7 Table 2: Key Publications from Studies of Cold Water Urchins Since 2000 2 5 Table 3: Key Publications from Studies of Warm Water Urchins Since 2000 2 6 Table 4: TP Mean and Standard Deviations After 120 Minutes In Vitro 5 6 Table 5: TP Mean and Standard Deviations After 120 Mi nutes In Vitro 5 6 Table 6: Comparisons of Median TPs After 120 Minutes In Vitro 5 7
1 Chapter 1 Introduction Invertebrates are backboneless organisms that together comprise over 90% of all known extant animal species and are related to the vertebrates through a common deuterostome ancestor (Figure 1 ). The echinoderms are phyla of inver tebrates, sister to the chordates, which are known to share a number of immune functions with vertebrates, and have therefore been used to examine the evolution of immunity in higher phyla ( Cooper 2001 ; Fujita et al. 2004; Pancer et al. 2006 ; Litman & Coop er 2007 ) Recent s tudies of echinoids have revealed that their immune systems are more complex and sophisticated than previously surmised and that they share a number of functions with higher vertebrates ( Nair et al. 2005; Smith et al. 2006; Sodergren 200 6; Tahseen 2008; Smith et al. 2011)
2 Figure 1 : The Phylogeny of Extant Phyla and Classes. A phylogenetic tree showing the common deuterostome ancestry of vertebrates and invertebrates (drawn by author) Prior to studies o f allograft transplants in the sea urchin Lytechinus pictus, urchins were not thought to possess an immune system. However, these studies indicated that urchins not only had an immune system, but that their immune
3 responses were more rapid and involved a g reater number immune effector cells than o ther known invertebrate s of the time (Coffaro and Hinegardner 1977) When t he urchin anatomy is considered this finding is not surprising : u rchins have a simple b ody plan and an inflexible test that renders them incapable of closing wounds via muscular contractions (Figure 2) (Chia 1996; Pancer et al. 1999) Therefore, u rchins have evolved highly efficie nt immune mec hanisms to repair damage to the test quickly and to prevent the flow of fluids into the external environment (Smith and Davidson 1994) This high degree of immune efficiency is essential for urchin survival within th e pathogen rich sea beds of their natural habitat, and may also contribute to their ability to live over a century (Pancer et al. 1999; Sodergren 2006 Smith et al. 2011 ) Figure 2 : The U rchin A natomy. Figure 2a. A cross sectional diagram illustrating the internal urchin anatomy. Note the location of the gonads and digestive tract (stomach and intestine). Figure 2b. Illustration of the oral surface of the urchin, no te the presence of the peristomial membrane and peristomial gills (from Wallace and Taylor 2002 )
4 In mammalian systems, leukocytes are the primary responders of the non adaptive immune response; in urchins and other echinoid species coelomocytes are the primary responders ( Parihar et al. 2010; Smith et al. 2011 ) In both mammalian and urchin systems, the immune cells involved in the initial defense response are non specific, non adaptive, and phagocytotic ( Fig ure 3 ) (Smith et. al 1995 ; Qudsia 2009 ) The phagocytotic cells of echinoids and higher vertebrates also share similar morphologies (Figure 4 ). Figure 3 : Compariso n of Mammalian and Echinoid Immune Cells (diagram modified from Smith et al. 1995) A vein diagram illustrating the similarities and differences between mammalian and echinoid innate immune cells. Note that the functions of the cells are highly similar.
5 Figure 4 : Comparison of a Phagocytotic Coelomocyte to a Human Macrophage. Figure 4 a. A n SEM of a phagocytotic coelomocyte from the sea urchin S. droebachiensis (image modified from Bertheussen and Seljeid 1978) X3000 Figure 4 b An ESEM photograph of a human macrophage (image modified from the Department of Physics Cavendish Laboratory ) 3420X The numerous similarities between the mammalian macrophages and echinoid coelomocytes which mediate host defense have long quali fied echinoid species as an appropriat e model for vertebrate immunity. In comparison to higher vertebrates, e chinoid anatomy is simple they can be easily maintained in captivity, they are diverse, and they inhabit a vast portion of the Ech inoid species such as the variegated sea urchin Lytechinus variegatus are therefore excellent candidates for the study of immune function and can be used to probe for conserved immune mechanisms within invertebrate and vertebrate immunity. I. An Introducti on to the Coelomocyte Coelomocytes are a class of mammalian like immune cells that are found free floating within the fluid filled body cavities of many invertebrate phyla including: echinoderms, mollusks annelids, nematodes, and arthropods
6 (Ravindranath 1980 ; Qudsia 2009; Ramirez Gomez & Garca Arrars 2010 ) Coelomocytes play an important role in the innate immune functions of their host organism and have been implicated in a wide array o f non specific defense mechanisms such as the formation of cellular clots, response to injury and infection, clearance of bacteria and foreign matter from the coelomic cavity, and the mediation of allograft rejection (Table 1 ) (Coffaro and Hinegardner 1977; Gerardi et al. 1990;
7 Table 1: Summary of Coelomocyte Morphologies and Functions (modified from Smith et al. 20 11 ) Cell type: % in WCF Morphology: Functions: Discussed by (Primary Author, Year): Phagocyte ** 40 78%* Amoeboid; diameter slightly acidophilic; contain intra nuclear iron bodies; may be in petaloid or filopodial forms. Ch emotaxis & cell activation Smith 1995; Chia 1996; Pancer 1999; Gross 2000; Borges 2005 Phagocytosis (Table 3*) Particle elimination Gerardi 1990; Ito 1992; Matranga 2005 Clotting Hillier 2003; Borges 2005; Smith 2006 ; Hillier 2007; Smith 2011 Encapsulation Johnson 1969; Isaeva 1990; Chia 1996; Smith 2006 Opsonisation Ito 1992; Gross 2000; Clow 2004 Graft rejection Edds 1993; Smith 2006 Red Amoebocyte 6 16%* Amoeboid; diameter averages contain many red spherical inclusions, which contain the pigment Echinochrome A. Chemotaxis Johnson 1969; Gross 1999 Antibacterial activity Johnson 1969; Haug 2002; Smith 2006 Degranulation Johnson 1969; Gross 1999 Blebbing D' Andrea Winslow 2008 O 2 Transport D' Andrea Winslow 2008 Colorless Amoebocyte 7 17%* Amoeboid; diameter averages contain many colorless spherical inclusions. Cytotoxicity Arizza 2007 Clotting? Smith 2011 Vibratile 13 29%* Highly mobile; diameter averages colorless cells; possessing a flagellum. Movement of WCF ? Bertheussen 1978; Smith 2006; Xing 2008 Clotting & secretion? Bertheussen 1978; Smith 2006 Values are sp ecific for L. variegatu s ; percentages of the cells within the coelomic fluid were combined from many reports from L. variegatus **Note that there are three subpopulations of phagocytes Invertebrate coelomocytes share a number of morphofunctional char acteristics with the immune cells of vertebrates (Aderem and Underhill 1999; Burke 1999; Qudsia 2009; Smith et al. 2001) The coelomocytes have therefore be en the focus of a large body of research focused on elucidat ing the mechanisms which
8 mediate immunity (Coffaro and Hinegardner 1977; Gross et al. 2000; Ma tranga et al. 2000; Smith and Davidson 1994) The mechanisms that underlay invertebrate immunity offer a wealth of information about the evolution of immunity and provide researchers with an alternative pathway to identify novel mechanisms that mediate im munity in vertebrates (Pancer et al. 1999; Rast et al. 2006) For these reasons, the coelomocytes have long been used as model systems for vertebrate immunity. Of the invertebrates, the echinoderms have been most extensively studied, with the vast majority of research focusing on sea urchins from the genus Strongylocentrotus Early studies of coelomocytes from the Atlantic purple sea urchin Strongylocentrotus purpuratus paved the way for studies whi ch focused on the composition of the coelomic fluid and various aspects of the coelomocytes within it (Johnson 1969; Johnson et al. 1970) Since the first observation and report of the coel omocytes by Bojanus in 1821, the cells have acquired numerous classification schema based on the morphofunctional characteristics of the cells (Bertheussen and Seljelid 1978; Kaneshiro a nd Karp 1980) It is universally agreed within the literature that the cellular composition of coelomic fluid is highly variable and is populated by a variety of morphologically distinct types of coelomocytes (Gross et al. 1999) In echinoderms, four subpopulations of coelomocytes are recognized by most authors, the phagocytes, red amoebocytes, colorless amoebocytes cells, and vibratile cells (Table 1 ). Confusion over the number of coelomocyte subpopulations has been further complicated by variations in coelomocyte populations even within the same species. For example, in male nematode Caenorhabditis elegans five types of
9 coelomocytes have been identified, while in hermaphrodites six are known to exist (Qudsia 2009) Due to dramatic variations in coelomocyte populations and densities across phyla, species, and between individuals, a universal classification system for these cells does not exist and coelomocytes are referred to b y numerous names even within current literature (see Ramirez Gomez & Garca review for more information on the populations of coelomocytes across various phyla ) For the purposes of this investigation, four subtypes of coelomocytes will be referenced and discussed: 1) the phagocytes (previously, phagocytotic amoebocytes ) ( 40 78 %), 2) vibratile cells (15 20%), 3) red amoebocytes (previously, red spherule cells ) (5 10%), and 4) colorless amoebocytes (previously, colorless spherule cells or mo rula cells ) (5 10%) (Table 1 ) (Bertheussen and Seljelid 1978; Borges et al. 2005; Johnson 1969; Matranga 2006) This terminology is in accorda nce with recent reports and observations made within the present study of coelomocytes from the Atlantic sea urchin Lytechinus variegatus (D'Andrea Winslow and Novitski 2008; Matranga 2006) II. Vibratile Cells Recent studies of vibratile cells have indicated that they are circular to oval in shape, and possess a long flagellar like extension, approximately 50 m in length, which protrudes from a basal granule (Xing et al. 2008) (Figure 5 ) In the sea urchin L. variegatus the cells have an approximate diameter of 7.5 m, and compose roughly 21% of the total coelomocytes within the perivisceral coelom (Mangiaterra
10 2001) The flagellar like extension of the vibratile cells allows the cells to rapidly travel in straight lines along a helical path throughout the coelomic fluid (Matra nga 2006 ) nt in samples of coelomic fluid in vitro initially led authors to conclude that vibratile cells both mixed and redistributed coelomic elements within the coelom in vivo (Bertheussen and Seljelid 1978) Later s tudies of vibratile cells in hanging drops indicated that the cells were neither efficient mixers of coelomic fluid nor did they play part in the distribution of coelomic elements throughout the fluid (Johnson et al. 1970) It is currently thought that the combined effect of the rapid movements of the vibratile cells, ciliated movements of the coelomic epit helium, and contractions of organs with in the coelom are responsible for the circulation of coelomic fluid in vi vo (Smith et al. 2006; Xing et al. 2008) Figure 5 : Image of a Vibratile C ell. Scale bar is 5 microns (image from Smith et al. 2011). Image captured by R. Bonaventura. The vibratile cells have also been implicated in clotting Betheussen and Seljeid (1978) captured light micrographs of vibratile cells that showed the cells are iately upon release. The authors also found that within fractions prepared from whole
1 1 coelomic fluid and suspended in anticoagulant, fractions containing higher numbers of vibratile cells would coagulate despite the presence of anticoagulant. These observa tions led the authors to conclude that upon contact with injured tissues, the cells were induced to release their mucoid containing granules, causing the fluid medium to coa gulate, and therefore initiate the process of clotting. These early studies of the vibratile cells form the majority of data available for their functions to date; no further evidence has suggested that the vibratile cells function in clotting. Recently, Xing and colleagues ( 2008) reported that the number of vibratile cells from the sea cucumber Apostichopus japonicus increased with repeated handling of the organisms. This observation is in agreement with a recent body of work that has focused on the activity of coelomocytes in response to stress (Bttger and McClintock 2009; Matranga et al. 2000; Matranga et al. 2002; Matranga et al. 2005; Matranga 2006) Recent studies of the coelomocytes have concentrated on the immune functions of echinoid coelomocytes as a whole, and apparently no new studies have focused on the function of vibratile cells or the gelatinous substance that is responsible coagulation (De Faria 2008; Hibino et al. 2006; Qudsia 2009; Rast et al. 2006; Smith et al. 2006; Sodergren 2006) III. Amoebocytes Red and colorless amoebocytes have been the center of a consi derable amount research in comparison to the vibratile cells (Sturycz and D'Andrea Winslow 2005) Initially, the amoebocytes were described as round or ovoid in shape however, it is now known that these cells are hi ghly mobile amoebocytes, and
12 their shape is constantly deformed as result of continuous blebbing (Figure 6) (D'Andrea Winslow and Novitski 2008) In fact, until quite recently these amoeboid cells (both red an d colorless) were named spherule cells due to their granular appearance and circular shape (Hetzel 1963; Isaeva and Korenbaum 1990; Smith and Davidson 1994) The use of this n omenclature has since been attributed to the anticoagulants and fixing media used during initial studies of the se cells. Matranga and colleagues (2006) have speculated that th e use of EDTA containing media such as anticoagulant solutions caused a compl which rendered the cells immobile and spherically shaped, leading to inaccurate descriptions of the cells. Time lapse recordings of amoebocytes from the sea urchin P. lividus revealed that the cells move through cyto plasmic extension of the leading edge (or possibly via a blebbing mechanism) and subsequent movement of the nucleus, at a rate of approximately 0.5 m/s (D'Andrea Winslow and Novitski 2008) Figure 6 : Images of Amoebocytes. Figure 6 a. A live red amoebocyte, image captured under light microscope Figu re 6 b. A live colorless amoebocytes, image captured under light microscope Scale bars are five mircrons (image s from Smith et al. 2 011). Images captured by R. Bonaventura.
13 There are two types of amoebocytes within the L variegatus : red and colorless. Both are similar in size and motility and their cytoplasm is densely packed with granules, which range from 0.2 to 5 m in diameter, that give the cells a morula like appearance (Chia 1996; Xing et al. 2008) Red amoebocytes are named for the red gra nules that fill their cytoplasm, which are significantly denser tha n the granules of colorless spherule cells, and can be separated from red granules using density centrifugation (D'Andrea Winslow and Novitski 2008; Smith et al. 2006) Red granules contain large quantities of echinochrome A, a napthoquinone pigment that gives the granules their red color and high density. Echinochrome A is known to have antibacterial activity against both gram negative and gram positive bacteria Treatment of echinochrome A against both gram negative and gram positive bacteria (Haug 2002) This is consistent with reports from studies of the sea urchin Echinus esculentus where coelomocyte derived echinochrome A has been found paired with coelomocyte proteins (Wardlaw and Unkles 1978; Wardlaw 1984) Taken together, the data suggest that the bactericidal activity of echinochrome is dependent on the integrity of an associated protein component that may be present within the coelomic fluid. Echinochro mes are also found in the tests of sea urchins (Kuwahara 2010) In urchin tests, echinochromes promote wound healing by acting as free radical scavengers which eliminate products formed by the autoxidation of tissue s at sites of damage (Lebedev 2005) In vitro red amoebocytes migrate toward bacteria, line
14 up, and form a wall to prevent further spread of bacteria (Johnson 1969) In vi vo red amoebocytes have been observed to migrate toward sites of compromised ti ssues and form a black, ring that surrounds the edge of damaged tissues (Gross et al. 1999; Johnson 1969) Red amo ebocytes have also been documented to localize at broken, infected, and regenerating spines in S. purpuratus In S. intermedius red amoebocytes were observed as red spots that manifested around an encysted larval parasitic worm within gonadal tissues (Shimizu 1994) Despite the l arge amount of research that has focused on the functions of the amoebocytes, l ess is known about the function of the colorless amoebocytes. Colorless amoebocytes have recently been shown t o have cytolytic activity that is calcium dependent ( Arizza et al. 2007 ). In their 2007 study, Arizza and colleagues found that the cytolytic activity of colorless amoebocytes was augmented by the presence of, or lysates from, red amoebocytes. This evidenc e suggests a highly cooperative relationship not only between colorless and red amoebocytes in host defense but between all coelomocytes within the coelomic fluid. This is further supported by the finding that proteins from the granules of both red and c olorless amoebocytes are also present in the coelomic fluid (Matranga et al. 2005; Matranga 2006) This evidence indicates that many of the soluble factors with in the coelomic fluid may be th e result of secretions f rom, or lysis of the amoebocytes (and by inference, the other coelomocytes) Taken together, these data indicate that the coelomocytes regulate the composition of the coelomic fluid by releasing soluble factors that signal to other cells within the coe lo mic fluid in response to host injury
15 or infection much like the release of cytokines from macrophages in mammalian immune reactions. IV. Phagocytes A. What is the phagocyte? A great body of work is dedicated to the study of the phagocytes due to the fundam ental role they play in echinoderm immune functions including chemotaxis, opsoninization, phagocytosis (Figure 7 a. ), clotting (Figure 7 b. ), encapsulation (Figure 7 c.), graft rejection, and the secretion of humoral factors (Gross et al. 1999; Smith et al. 2006) Phagocytes are also the main expressers of immune response genes including complement proteins, a C type lectin, and transcription factors (Clow et al. 2004; Gross et al. 1999; Pancer et al. 19 99; Smith et al. 1998) .The high level of immune genes expressed by urchin phagocytes emphasize that the cells are a crucial component of host defense. F igure 7 : Immune Functions of Phagocytes. Phase contrast images of the phagocytes engaged in various cellular activities. Figure 7 a. (from Adamowicz and Wojtaszek 2001) A phagocyte with a n ingested yeast cell ( arrow ), 945X Figure 7 b. ( from Hillier and Vacquier 2003 ) a group of phagocytes forming a cellular clot. Bar 15 m. Figure 7 c. (from Bertheu ssen and Seljelid 1978) A brown body; the result of encapsulation, 500X.
16 There are three subpopulations of phagocytes: polygonal, discoidal, and small (Brockton 2008; Smith et al. 2006) Each can be distinguished from another by their respective morphology, density, and differential expression of SpC3. Polygonal and discoidal phagocytes express greater quantities of SpC3 than the small phagocytes in S. purpuratus and preliminary observations o f the small phagocytes in vitro have indicated that the cells may initiate the formation of syncytia (Bertheussen and Seljelid 1978) The differential expression and morphologies of the phagocytes led authors to conclude that there are three subpopulations of phagocytes (small phagocytes were previously thought to be a less mature form of the larger discoidal and polygonal phagocytes) and indicate that each may serve a different function in host defense (Brockton 2008; Gross et al. Smith 2000) Phagocytes are the only coelomocytes known to contain intra nuclear (ferritin) iron bodies, and the cells can be found throughout the coelomic fluid and connect ive tissues (Borges et al. 2005; Hillier and Vacquier 2003; Smith et al. 2006) In L. variegatus, phagocytes comprise from 40 78% of the coelomocyte population, and their n umbers within the coelomic fluid vary according to the individual sampled and the site from which coelomic fluid is removed (Borges et al. 2005; Mangiaterra 2001) B. Petaloid vs. Filop odial When stressed by removal from an organism, or when in contact with foreign matter, all three subpopulations of phagocytes will undergo a transformation from the petaloid to filopodial form (Figure 8 ) (Edds 1993; Gross et al. 1999; Johnson
17 1969; Smith et al. 2006) Small phagocytes can be distinguished from discoidal and polygonal phagocytes on glass slides because the small phagocytes do not spread onto glass as extensively as discoidal and polygonal populations (Brockton 2008) Both petaloid and filopodial phagocytes have pseudopodia that radiate in all directions from the central perinuclear cytoplasm (Xing et al. 2008) but what distinguishes between the two is the lame l lipodia that edge them. In the petaloid form, the phagocytes have bladder like petals that extend in all directions from the central region of the cell (Figure 8 a. ) (Chia 1996; Smith 1981) and upon contact with glass, a foreign target, or when suspended in hypotonic media, petaloid phagocytes will transform into filopodial morphology. This transformation occurs as serrations at the lamellipodial edge begin to form and cytoplasm is retracted from the cell edge; the transformation is complete when filopodia are extended (Chia 1996; Smith and Davidson 1994) Figure 8 : Petaloid and Filopodial Phagocytes In vitro (f rom Xing 1998) Phagocytes with petaloid (left) and filopodial (right) morphologies. Figure 8 a. Petaloid phagocyt es before p hagocytosis. Figure 8 b. Filopodial phagocytes after phagocytosing latex beads.
18 The phagocytes transformation from petaloid to filopodial form heads the clotting r esponse to injury and the elimination of foreign invaders Like the colorless amoebocytes se nsitivity to red amoebocytes and their lystates, the phagocytes sensitivity to changes in coelomic fluid solute concentrations point to a dynamic relationship between the phagocytes and the coelomic fluid; wherein the coelomocytes and coelomic fluid are pa rt of a signaling cascade that mediates host defense C. Clotting Clotting is an extremely important component of the urchin defense response because contrary to other invertebrates (such as the earthworm Lumbricus terrestris ) and vertebrates, urchins are n ot capable of aiding wound closure via muscular contraction (Figure 9 ) (Smith and Davidson 1994) Phagocyte clotting allows for urchins to stop the flow of coelomic fluid into the surrounding environment when the tes t is punctured. All three populations of c oelomocyte s are known to form cellular clots (Borges 2002 ; Borges et al. 2005 ) It is thought that upon injury to the urchin test, vibratile cells loca lize to the site of injury, and degranulate releasing a substance that gels immediately upon its release. Following the localization of the vibratile cells to the site of injury, phagocytes within adjacent connective tissues and the coelomic fluid are ac tivated, and begin to form a cellular clot ( Hillier and Vacquier 2003; Xing et al. 2008)
19 Figure 9 : Urchin Anatomy vs. L. terrestris Anatomy (modified from Wallace and Taylor 2002; Wallace and Taylor 2002) Urchins lack the musculature found within many invertebrate and vertebrate species, therefore they have evolved a highly effici ent clotting mecha nism. Figure 9 a. The gene ralized urchin anatomy. Figure 9 b. The generalized anatomy of the earthworm L. terrestris Compare the musculature, which lines the inner body wall, of L. terrestris to the lack of urchin musculature beneath the test. I t has be en shown that intracellular coelomocyte clotting is mediated by an olfacto medin (OLF) protein called A massin 1 (Hillier and Vacquier 2003) Amassin 1 which is a coelomic plasma protein, is the first OLF protein fo r which a function has been elucidated. The protein participates in phagocyte adhesion by forming large disulfide bonded aggregates between phagocytes. Structural analysis of the Amassin 1 is simila r to vertebra te proteins of unknown function, indicating that OLF proteins may also function in cell cell adhesion in vertebrates (Clow et al. 2004; Hillier and Vacquier 2003) Later studies by Hillier and co lleagues revealed that Amassin 1 is one of five OLF genes expressed by sea urchins; four of which are a sub group of previously identified OLFS, now known as the amassins, and the last is an OLF within the colmedin sub family whose functions are currently unknown (Hillier et al. 2007) This study also revealed that the OLF coding genes are highly transcriptionally regulated during
20 urchin larval development which likely has implications for the role of colmedins in both urchin and ver t ebrate development. Clotting is also central to the encapsulation of foreign matter that is too large for a single phagocyte to consume. During the process of encapsulation, phagocytes suspended within the coelomic fluid come in contact with one another, begin to adhere, and form a large sync y tium to surround the invader (Isaeva and Korenbaum 1990; Smith et al. 2006) Several particles can be caught within a single clot (current study Figure 18 b. ). As phagocytes adhere to one another, nearby vibratile cells and amoebocytes are passively caught within in the clot. Red amoebocytes caught within the capsule degranulate, release echinochrome A, and degrade the trapped entity (Gross et al. 1999; Johnson 1969) The end products of encapsulation have been termed brown bodies, which have been shown to vary in size and shape (Hetzel 1965) B rown bodies are colored by melanin, a pigment released from the granules of amoebocytes caught within the capsules (Chia 1996) Until recently, it was thought that once amoebocytes were caught within a phagocyte clot they remained trapped. However, Matran ga and colleagues (2006) have used time lapse imaging of clots in vitro to show that amoebocytes only remain trapped within cellular clots briefly, and that the cells usually escape from the clots. These clots in vitro are characterized by a white to reddi sh mass of cells (De Faria 2008) D. Chemotaxis Chemotaxis, or the directional migration of cells in response to a chemical gradient, is an essential component of the immune response of urchins. Red
21 amoebocytes and phagocytes have been shown to migrate toward bacterial stimuli both in vivo and in vitro (De Faria 2008; Mangiaterra 2001; Pancer et al. 1999; Smith and Davidson 1994) Coelomocytes can migrate toward many chemical stimuli that vertebrate macrophages also migrate toward. Amoeboid coelomocytes, such as red and colorless amoebocytes and phagocytes, can be induced to directionally migrate toward bacteri al components such as lipopolysacharride (LPS), phormil, methyl, leucil phenylanine (fMLP), mammalian cytokines, and tumoral nueropeptides (Borges et al. 2005) Chemotaxis is induced when a chemoattractant bind s a surface receptor embedded in the membrane of a mobile cell. Once bound to a cell surface receptor, a signaling pathway is activated, and actin is actively polymerized at one pole of the cell, which becomes the pseudopod (Chung 2001) In S. purpuratus amoeboid coelomocytes have been shown to express profilin, a n actin binding protein which is known to stimulate cellular activation by sequestering actin monomers to growing actin monofilaments (that are essential fo r cytoskeletal restructuring) (Carlsson et al. 1977; Cooper 1991 ) P rofilin also has a greater affinity for ATP than actin monomers, which catalyzes the phosphorylation of actin bound ADP to ATP and subsequently the growth of monofilaments at the leading e dge of an activated cell (Gross et al. 1999; Pancer et al. 1999 ; Kovar 2006 ) S tudies of urchins injected with lipopolysacharride (LPS), a major component in gram negative bacteria, reported that c oelomocytes greatly enhanced profilin gene transcription along with a host of other immune genes notably involved in RNA splicing, protein recognition, cell signaling, and alterations to the cytoskeleton (Smith et al. 1995 Nair et al. 2005 )
22 These data ar e in agreement with the activation of macrophage s in higher vertebrates, wherein macrophages are activated upon recognition of foreign ligands, and chemotax toward higher concentrations of the chemoattractant via cytoskeletal rearrangement (Chung 2001 Mosser & Edwards 2008 ) E. Phagocytosis The phagocytes have been extensively studied with respect to phagocytosis ( see in Table 3 ) Urchins eliminate foreign particles from the coelomic fluid via phagocytosis and enc apsulation (Ito et al. Following activation by chemotactic factors, petaloid phagocytes move toward and bind to chemoattractants such as damaged cells, microbial products, and complements (r eviewed by Chung 2001 ) Target cells are opsonized by proteins in the coelomic fluid which enhance adhesion of phagocytes to their targets and increase the rate of phagocytosis (Clow et al. 2004; Gross et al. 2000; Zhu et al. 2004) Once a petaloid phagocyte has bound its target, it transforms into the filopodial form, and extends filopodia to engulf the target cell in a phagosome (Chia 1996) Wh en the target has been fully engulfed, it is degraded by lysosomal enzymes and hydrogen peroxide in (Matranga et al. 2005; Smith et al. 2006) Following phagocytosis, phag ocytes in a variety of organisms have been observed to migrate to a number of organs including gills, respiratory trees, and axial organ from where they are secreted to the external environment for elimination (Ito et al. 1992; Smith 1981)
23 Only large phagocytes in the petaloid form have been observed to uptake particles (Borges 2002; Clow et al. 2004; Matranga et al. 2005) The phagocytes are highly efficient at recognizing bacteria, cellular debris, foreign cells, and inert particles (Chia 1996) Hydrogen peroxide, reactive oxygen species, and lysosomal enzymes are essential for the d egradation of foreign particles in mammalian phagocytes (Aderem and Underhill 1999) Similarly, Ito and others (1992) found that phagocytes from the urchin S. nudus produce hydrogen peroxide continuously, and that hyd rogen peroxide production increases when phagocytes are activated. This study and others with urchin coelomocytes also reported that the rate of particle ingestion could be increased if the target cells were first incubated (or opsonized) in coelomic fluid (Gross et al. 2000; Haug 2002; Ito et al. 1992) Opsonization is central to the elimination of foreign invaders in urchins and mammalian systems (Al Sharif et al. 1998; Pancer et al. 1999; Smith et al. 1998; Smith et al. 1999; Smith et al. 2001; Sodergren 2006) Opsonins and a variety of hu moral factors that function similarly to immunoglobins (including lectins, agglutinins, and lysins) can be found in the coelomic fluid of echinoderms (Chia 1996 Kudriavtsev & Polevshchikov 2004 ) Opsonins augment th e rate of phagocytosis by coating the foreign target with proteins that allow phagocytes to more rapidly recognize and tightly bind to foreign invaders. Clow and colleagues (2004) identified SpC3, a homologue of complement component protein C3, from S. pur puratus SpC3 was shown to be a protein that acted as an inducible humoral opsonin; w hen yeast were incubated with SpC3, it was found that petaloid phagocytes consumed the yeast with greater efficiency. These data indicate that
24 SpC3 most likely binds to a type 3 complement receptor emb edded in the phagocyte membrane, and that it is likely that sea urchins have a complement system similar to that found within higher vertebrates (Bertheussen and Seljelid 1978; Smith et al. 2006) F. Lytechinus variegatus The similarities between mechanisms that operate in echinoderm and mammalian immunity have been the foundation for numerous studies of urchin immunity. The majority of this research has focused on urchins found in colder waters such as the Atlantic and Antarctic (Table 2) Literature that discusses the immune functions of urchins from tropical waters, such as L variegatus is less extensive (Table 3 ) Of the research on L. variegatus the phagocyt es are most thoroughly considered (see *Table 3)
25 Table 2 : Key Publications from Studies of Cold Water Urchins S ince 2000 Primary Author Year of Publication Species Major Conclusion Gross 2000 S. purpuratus SpC3, a homologue of the human comple ment component C3, is expressed by phagocytes. Pancer 2000 S.purpuratus Coelomocytes express molecules with a high degree of genomic and transcriptional complexity. Borges 2002 S. neumayeri Rates of phagocytosis in S. neumayeri are lower than in tropical urchins. Brooks 2002 S. purpuratus & L. variegatus The major yolk binding protein in urchins may shuttle iron to developing germ cells. Haug 2002 S. droebachiensis Coelomocytes and body wall extracts have the high antimicrobial activity. Hillier 200 3 S. purpuratus Amassin, a novel coelomic plasma protein, is responsible for phagocyte clotting. Clow 2004 S.purpuratus Phagocytes have an SpC3 receptor that mediates phagocytosis. Rast 2006 S. purpuratus Urchins share a number of cell surface receptor s and genes that are expressed within vertebrates. Smith 2006 S. purpuratus The cornerstones of urchin immunity are opsonization and phagocytosis. Sodergren 2006 S.purpuratus The first authors to sequence and analyze the 814 megabase genome of S. purpu ratus. Brockton 2008 S. purpuratus Small and polygonal phagocytes upregulate 185/333 proteins in response to immune challenges. Li 2008 S. droebachiensis Strongylocin 1 & 2, antimicrobial peptides (AMPs) expressed in coelomocytes, have antimicrobial act ivity. Borges 2010 S. neumayeri Intra nuclear iron bodies increased in the phagocytes of urchins when exposed to water soluble fractions of oil.
26 Table 3 : Key Publications from Studies of Warm Water Urchins since 2000 Primary Author Y ear of Publication Species Major Conclusion Silva 2000 L. variegatus Phagocytosis was first detected during the mid gastrula stage of urchin embryo development. Mangiaterra 2001 L. variegatus Phagocytes mediate the urchin inflammatory response. Bro oks 2002 L. variegatus & S. purpuratus The major yolk binding protein in urchins may shuttle iron to developing germ cells. Borges 2005 L. variegatus Phagocytes in oral regions have greater phagocytotic activity and more intra nuclear iron bodies than phagocytes in aboral regions. D'Andrea Winsolw 2008 L. variegatus Blebbing is controlled by an acto myosin contractile mechanism in red and colorless amoebocytes. Powell 2008 L. variegatus Urchin diet affects coelomocyte populations and potentially immune functions. Bottger 2009 L. variegatus Organic phosphate exposure sever e ly impairs bacterial clearance from WCF; inorganic phosphate exposure also impairs bacterial clearance. I ndicates that the main findings from this study are closely relat ed to phagocytes or phagocytosis. In the variegated urchin L. variegatus the total coelomocyte population was found to be 2 2.7 x 10 3 cells/mL whole coelomic fluid (WCF) (Borges et al. 2005) Comparative studies of coelomocyte populations in oral and aboral regions of the perivisceral coelom of urchins maintained in a mouth down position for 24 hours found that cell density was greater in oral regions (2.752 x 10 3 cells/mL) than aboral regi ons (2.007 x10 3 cells/mL). When urchins were kept mouth up, coelomocyte cell density was still observed to be higher in oral regions (3.061 x 10 3 cells/mL) than aboral regions (2.192 x 10 3 cells/mL). The phagocytes were found to comprise 63 77% of the total coelomocyte population, and their density was greater in the oral region (2.016 x 10 3 cells/mL) than in the aboral region (1.2705 x 10 3 cells/mL) (Borges et al. 2005) Borges also found that phagocytes removed fro m
27 the oral region had a higher percentage of intra nuclear iron crystals and a higher capacity for phagocytosis. Mangiaterra and Silva (2001) used heat sterilized yeast to induce an inflammatory response within L. variegatus. They reported that ferritin l abeled phagocytes were activated when India ink or sterile yeast were injected into the peristomial connective tissues, and were then induced to migrate through the connective tissues toward the site of injection. Once at the site of injection, the phagocy tes were observed to phagocytose the injected India ink and yeast Similar experiments with the Brazilian sea urchin Echinometra lucunter found that only phagocytes were capable of endocytosing ferritin from the coelomic fluid in vivo (De Faria 2008) Phagocytes that ingested ferritin from the coelom were used as markers for phagocytes that had previously resided within the coelom. Ferritin labeled phagocytes were observed to actively migrate into peristomial conn ective tissue when it was injected with India ink. The authors concluded that the phagocytes activity in response to India ink injection could be termed an innate immune response, and that the phagocytes were the mediators of infection in E. lucunter In vitro studies of the clearance rates of the marine bacterium Vibrio splendidus from the coelomic fluid of L. variegatus reported similar findings to the clearance rates of V. anguillarum from the sea urchins S. purpuratus and S. droebachiensis In L. variegatus the greatest amount of bacterial clearance (90 90% of total bacteria) occurred within
28 the first 24 hours of exposure (Bttger and McClintock 2009) Higher clearance rates within the first 24 hours of bacterial exposure have also been reported in other sea urchins (Borges 2002; Haug 2002) Humoral secreti ons such as cytolytic, bactericidal, and agglutinating factors contained within the coelomic fluid are thought to play an important role in bacterial clearance (Chia 1996; Gross et al. 1999) Howev er, bacterial clearance rates in L. variegatus did not show significant increases when bacteria were exposed to coelomocyte free coelomic fluid before assayed (Bttger and McClintock 2009) Environmental conditi ons such as temperature and salinity are known to affect urchin activity. Lawrence showed that adult L. variegatus show the greatest righting activity when water conditions were at 28 C and 35% osmolarity (Lawrence 1975) It is also thought that environmental conditions affect coelomocyte populations (Matranga et al. 2000; Matranga et al. 2005; Powell et al. 2008) Likewise it may be possible that coelomocytes function with optimal activity in certain conditions, although no literature has been found to support this notion. It has been suggested within the literature that diet can affect the population of coelomocyte subtypes in urchin s (Mangiaterra 2001; Qudsia 2009; Smith et al. 2006) T he correlation between coelomocyte subpopulation density and urchin diet in L. variegatus is under investigation, alt hough no conclusive evidence has been reported (Powell et al. 2008) In light of the effect of external conditions on coelomocyte population, recent studies have investigated the effect of polluted and physically s tressed
29 environments on the coelomocytes (Matranga et al. 2000; Matranga et al. 2002; Matranga et al. 2005; Matranga 2006) Chronic exposure of L. varieg atus to sub lethal concentrations of inorganic (sodium) and organic (triethyl) phosphate was found to profoundly decrease the rate of bacteria clearance from coelomic fluid. The extent of reduction in the rate of bacterial clearance was dependent on the ty pe of pollutant used: inorganic exposure lowered bacterial clearance rates, while organic exposure completely impaired the phagocytes ability to eliminate bacteria over a four week span (Bttger and McClintock 2009 ) These studies with L. variegatus and other urchins have indicated that it may be possible to use urchin coelomocytes as a biosensor for ocean health (Bttger and McClin tock 2009; Matranga et al. 2005; Matranga 2006) Many mechanisms that regulate particle recognition and phagocytosis in invertebrate systems are conserved among mammalian systems. Phagocytosis is known to be a complex process mediated by cell surface re ceptors such as Fc receptors, C 3 receptors, and foreign surface receptors (Aderem and Underhill 1999; Bertheussen and Seljelid 1978) The regulation of phagocytosis is essential for inflamm ation and homeostasis functionality. Despite the great body of work dedicated to the mechanisms that underlay phagocytosis in other invertebrates, little work focused on such regulatory mechanisms in L. variegatus (Adamowicz and Wojtaszek 2001; Borges 2002; Burke 1999; De Faria 2008; Peiser et al. 2002; Smith et al. 2006)
30 Studies of coelomocytes from the earthworm, Lumbricus terrestris showed that phagocytes responded via directional migration to bacterial and foreign tissue in vitro (Marks et al. 1979) Interestingly, it was found that when L. terrestris phagocytes were added to assay wells containing L. terrestris body wall and foreign tissues findings led t he authors to conclude that L. terrestris body wall contained a migration inhibitor. It does not appea r that evidence has since been reported to support or refute this finding. D irectional migration toward a chemical signal (chemotaxis) precedes particle engulfment (phagocytosis), therefore it was conjectured that phagocytosis in L. terrestris may also be affected by such an inhibitor. Both L. terrestris and L. variegatus possess a true coelom, are linked by a common deuterostome ancest o r and are regulated by the same immune cells (coelomocytes); these shared features sparked interest in whether L. vari egatus tissue s may similarly contain an inhibitor of phagocyte migration and possibly phagocytosis. T his study was generated to determine if the presence of L. variegatus tissues (digestive tract, gonad, peristomial gill, and peristomial membrane) have an inhibitory a ffect on inflammation (herein characterized a s a response regulated by the phagocytotic coelomocytes ) in L. variegatus To evaluate inflammation in L. variegatus t wo central questions w ere posed and examined using in vivo and in vitro assay s: f irstly, does the presence of yeast cause an inflammatory response, as characterized by a significant change in the median number of total phagocytes
31 ( T P), in L. variegatus ? S econdly, does the presence of L. variegatus tissue cause the efficiency with w hich phagocytes consume yeast (PI) to deviate from their PI in the absence of L. variegatus tissues ? If there was n ot a significant deviation from the control median TP in response to the presence of yeast, it was assumed that the presence of yeast d oes not illicit an inflammatory response as mediated by L. variegatus phagocytes Conversely, if the presence of yeast did cause a significant deviation from the control median TP, it was assumed that the presence of yeast does elicit an inflammatory response in L. variegatus I f there was not a significant deviation from the control PI when L. variegatus phagocytes were in the presence of both yeast and L. variegatus tissue, it was assumed that the presence of L. variegatus tissues has no affect on the regulat ion of phagocytosis in L. variegatus Conversely, if the presence of L. variegatus tissues did cause a significant deviation from the PI of control urchins, it was assumed that the presence of L. variegatus tissues did have a regulatory role in the phagocy tosis of yeast in L. variegatus
32 Chapter 2 Methods This study was generated to determine if the phagocytotic capacity and index of phagocytes from L. variegatus is affected by the presence of L. variegatus tissues. A previous study of coelomocytes f rom L. terrestris reported that the migratory media containing L. terrestris body wall. Due to common ancestry and the presence of coelomocytes in both L. terrestris and L. variegatus, it was therefore suspected that tissues from L. variegatus may similarly affect L. variegatus phagocytes. The finding that the migration of L. terrestris phagocytes were impaired by the presence of L. terrestris body wall suggested that phagocy tosis may also similarly be impaired. Therefore, the effect s of L. variegatus tissue on the phagocytotic index and capacity of L. variegatus phagocy tes were determined as described below. I. General A. Ur chin Collection and Maintenance Sea urchins ( Lytechinus variegatus ) were collected from the turtle grass ( Syringodium filiforme and Thalassia testudinum ) beds of Ken Thompson Parkway in Sarasota, Florida ( Figure 10 ) Animals that were approximately 7cm in diameter or larger were kept, transported in a cooler ba ck to Pritzker Marine Laboratory, equilibrated to tank temperature (21 22 C) via a slow drip, and maintained in a 550L tank containing running sea water (pH 7.4; 29 0 / 00 ). Sea water was pumped from the Sarasota Bay, filtered, and sterilized. The urchins wer e fed an omnivorous
33 diet (chopped shrimp, fish chunks, pellet, and raw spinach) three times weekly and were kept on a regular light cycle. Figure 10 : Urchin Collection Site. L ytechinus variegatus were collected from the tu rtle grass beds (indicated by yellow) of Ken Thompson Parkway, Sarasota, Florida (image modified from Google Maps). B. Removal of Coelomic Fluid: Whole coelomic fluid (WCF) (~pH 6.9) was withdrawn from the oral region of the peristomial coelom by puncturin g the peristomial membrane with a sterile BD inserted diagonally through the peristomial membrane to ensure that the oral region of peristomial coelom was intercepted with out puncturing other organs. Once the needle was inserted through the peristomial membrane, WCF was slowly and carefully withdrawn into syringes containing equal amounts of clot inhibiting medium (CIM) (Appendix 1) As WCF was withdrawn, syringes were gen tly rolled to help mix the WCF with CIM to minimize clotting.
34 Following removal, the WCF/CIM mixture was immediately transferred into sterile 2mL microcentrifuge tubes by slowly injecting the WCF/CIM mixture against the wall of the tube. Mircocentrifuge t ubes were then capped, gently rolled to ensure thorough mixing of WCF with CIM, labeled, and kept at room temperature (~23 C). C. Preparation of Heat Sterilized Yeast: T o prepare target cells for the phagocytosis assays, sterile stock solutions of east ( Saccharomyces cerevisiae yeast was placed into an Erlenmyer flask, suspended in filtered sea water (FSW, pH 6.8), and autoclaved at 121 C for 15 minutes. Once cooled to room temperature, the suspension was vigor ously swirled to resuspend cells, and then pushed through a times to de clump large cell aggregates. The resulting frothy mixture of yeast was then transferred into 2mL microc entrifuge tubes, centrifuged for 2mins at 600g, and washed with ~1.5mL FSW by three rounds of centrifugation for 2mins at 600g. Af ter washing, yeast were labeled and stored at room temperature (23 C). The density of the yeast in stock solutions was determ ined by diluting aliquots of stock with FSW and the cells were counted using a hemocytometer. Yeast suspensions of desired cell density were prepared by diluting stock solutions with aliquots FSW. i. Yeast Staining with Eosin Y Based on previous studies of c oelomocytes that used yeast as target cells in phagocytosis, yeast used for in vitro phagocytosis assays were stained with Eosin Y (Toupin et al. 1977) Eosin Y is a fluorescent red dye that stains the cytoplasm of
35 non viable cells red. Following washing with FSW, sterile yeast were suspended in 2mL 1% filtered Eosin Y solution, and then washed by six rounds of centrifugation for 2 mins at 600g (23 C). The stained yeast were then resuspended in 1mL FSW and kept at room temperature until later use in assays. ii. Preparation of Eosi n Y yeast to 1,008 yeast/mL FSW Yeast were adjusted to a final concentration of 1,008 yeast/mL for in vitro phagocytosis assays. Based on previous evidence that the population of L. variegatus pha gocytes was 2,016 phagocytes/mL WCF in oral regions, the population of oral phagocytes in equal portions of WCF/CIM was taken to be 1.008 phagocytes/mL WCF/CIM (Borges et al. 2005) Based on these numbers, yeast w ere then added to assay chambers such that the final concentration of yeast to phagocyte was 10:1. Coelomocytes from the oral region of Lytechinus variegatus have been experimentally determined to have a density of 2.752 x 10 3 cells/mL whole coelomic fluid (WCF), of which approximately three quarters (73.24%) of the WCF population are phagocytotic amoebocytes (2.016 x 10 3 PA/mL WCF) (Borges et al. 2005) Yeast suspensions used for phagocytosis assays (1:10 coe lomocytes per yeast) were prepared according to these numbers. D. Yeast Counting Cell density was determined using a Hausser Metallized Reichert Bright Line Hemocytometer Cell suspensions introduced into the chamber were prepared to concentrations dilute enough such that the cells did not overlap in the counting grid. Once loaded into the hemocytometer, cells were systematically counted within
36 selected squares, their numbers summed, averaged, and then used to extrapolate an estimate for total cell density. Cells were counted from twelve 1/400mm 2 squares, which lay within three 1/25mm 2 squares, across the diagonal of the counting grid using a compound light microscope Yeast cells which laid on the right or bottom lines of the counting squares were exclud ed from cell counts. Yeast cell aggregates were also excluded from the cell count ( Figure 11 ). Figure 11 : Diagram Illustrating Yeast Counts. To clarify yeast counting methods : Red squares correspond to 1mm, yellow to 1/25 m m 2 and green t o 1/400mm 2 Figure 11 a A diagram representing the counting grid of a hemocytometer. Yeast were counted from green squares Figure 11 b Numbered cells in the green bubble show yeast counted. indicates cells excluded from cellular count s. E. Determination of Coelomocyte Density Determining coelomocyte density using the method outline d above for yeast counts proved to be difficult. T he cells were often at such a low density that it was not possible to obtain a cellular count large enough to be considered significant (100+ cells). T he small amount of suspension that the counting chamber holds (10 L) rapidly dried under the heat of the light microscope. I t was difficult to
37 visualize the unstained cells. The use of stains on the hemocytometer was not optimal because any undissolved crystals of stain would catch in the loading well and impair the cel ls abilities to load over the counting grid. For these reasons, the density of L. variegatus coelomocytes was taken from Borges and others 2005. II. Experimental A. Observation of Coelomocytes In V itro Coelomocytes were observed in vitro to assess cellular mov ement, morphology, and chemotaxis. WCF was collected, prepared in wet mounts and hanging drops, and then observed under a compound light microscope. i. Observation of C oelomocytes in W et M ounts WCF was removed from urchins using the technique previously de scribed, dropped onto a clean slide, a cover slip was placed over the drop, and the slide was immediately observed by light microscopy. Several slides could be rapidly prepared in this manner and the four types of coelomocytes, red and white amoebocytes, phagocytes, and vibratile cells, could be visualized. Wet mounts could only be viewed for approximately 2 3 minutes before the slide dried from the heat of the light microscope. ii. Observation of C oelomocytes in H anging D rops Hanging drop slides u se an in verted droplet of solution, which allows for the solution to be observed for longer periods of time without drying under a light microscope than wet mount slides. Hanging drops were prepared from WCF/CIM by rapidly dropping one drip of WCF/CIM onto the cen ter of a clean cover slip and small mounds of Vaseline were then applied to each corner of the cover slip. A
38 depression slide was then inverted, carefully centered over the droplet on the cover slip, and pressed down gently to form a seal between the depre ssion slide and cover slip. The droplet between the depression slide and cover slip (now adhered) was then suspended by rapidly inverting the slide in a fluid motion. Hanging drops were then observed under a phase contrast light microscope for several minu tes at a time. B. Phagocytosis A ssays The phagocytotic activity of L. variegatus coelomocytes was assayed both in vivo and in vitro Sterile yeast suspended in pH adjusted FSW were used as target cells for coelomocyte phagocytosis. A single in vivo assay wa s performed by direct injection of yeast into the coelom; the study served as a control for the phagocytotic capabilities of coelomocytes in vivo Two types of in vitro assays were performed: one to determine a baseline for phagocytosis in vitro and the s econd to determine if L. variegatus tissues caused phagocytosis to deviate from the baseline determined for phagocytosis in vitro i. Experiment 1: In vivo phagocytosis assays using yeast in FSW Yeast suspensions were directly injected into the coelom o f nine urchins to assay the rate of yeast elimination in vivo Urchins were collected and kept in individual containers coated with a thin layer of Vaseline prior to experimentation. Two hundred microliters of heat sterilized yeast suspended in FSW (10 0 mg yeast/mL) was injected into the coelom through the peristomial membrane. The time of injection was recorded and a 15 minute incubation period was allowed container was lifted and gently circled for one minute to aid in the distribution of
39 injected yeast throughout the coelom and urchin activity was recorded at timed intervals throughout the 2hr assay. Urchin activity was recorded with respect to spine activity, tube feet activi ty, and crawling (up the sides of the container). At set intervals of 0mins, 30mins, 60mins, and 120mins (0mins refers to the first sample removal following incubation); approximately 250 L of WCF was withdrawn from each urchin into individual syringes preloaded with 250 L of CIM. Immediately upon removal, syringes were gently rolled to ensure WCF thoroughly mixed with CIM. Approximately 250 L of the WCF/CIM mixture was pushed through the syringe, collected, and pooled with samples removed from all individuals. Of the remaining WCF/CIM in the syringe (~250 L), three drops were placed on two slides and a thick smear was prepared using a separate sterile slide. Only one smear was prepared fo r control organisms. Smears were allowed to air dry (overnight), were fixed twice in 100% methanol for 5mins, rinsed with DI H 2 O, 2 O) for 30mins, then gently rinsed with DI H 2 O, and finally blotted dry with bibulous paper. Smears were then examined under a light microscope and cellular counts were performed. Two urchins were prepared, for each of two types of control conditions, and monitored during in vivo yeast assays. The first type of control was a true control lef t alone for the duration of the experiment except for when WCF was withdrawn The second type of control organism was a control for the effect of FSW, these urchins were inject ed with 200 L of FSW then left to incubate with the other urchins. Since the yeast used in all phagocytosis assays were suspended in FSW, it was necessary to examine if FSW affected the phagocyte population. WCF was
40 withdrawn from both controls as described for experimental organisms above, and a single smear was prepared and stain ed for each time interval. Figure 12 : Flow Chart for Experiment 1: In Vivo Phagocytosis Assay A flow chart illustrating the experimental design of in vivo phagocytosis assays. 1. Urchins were first observed in individ ual holding tanks and their activities recorded (with respect to tube feet activity, spine activity, and crawling). 2. Urchins were then injected with control and experimental suspensions using 1mL hypodermic needle. 3. Immediately following injection, a timer was set, and the urchins were allowed 15minute incubation period (wherein the urchins were left undisturbed). 4. Following incubation, 0.2mL of WCF was withdrawn from each urchin into syringes preloaded with 0.2mL of CIM at the timed intervals of 0mins, 30mins, 60mins, and 120mins; syringes were carefully rolled as WCF was removed to ensure thorough mixing. 5. The relative color intensity of the resulting WCF/CIM mixture was recorded on a scale from 1 (least inten se) to 3 (most intense). 6. Individual smears were then prepared using the WCF/CIM mixture from the urchins at each time interval the cells on each smear were counted and the TP and phagocytosis index (PI) determined.
41 ii. In V itro P hagocytosis A ssays Urchins were assayed for their ability to eliminate eosin Y stained yeast in both the absence and presence of urchin tissues in vitro a. Experiment 2: As says using E osin Y stained Y east Twelve urchins were assayed for their ability to eliminate eosin Y stained yeast from coelomic fluid in vitro Urchins were collected from their tanks, placed into temporary holding containers, labeled, and observed. Urch in physical activity was recorded with respect to spine movement, tube feet activity, and crawling up the side of the holding container. Urchins were then weighed: each specimen was held out of water for 15secs to remove excess water and examined for injur ies. Urchin injury was recorded with respect to missing and/or broken spines, physical abrasions to the test, and other abnormalities. Five hundred microliters of WCF were withdrawn from each urchin into 1mL sterile syringes, which were fitted with 20 gau with 500 L CIM. During WCF removal from the organism, the syringes were gently rolled to aid in mixing of the WCF and CIM. The resulting WCF/CIM mixture was then carefully and slowly pipetted down the side of labeled ste rile 2mL microcentrifuge tubes, tubes were capped, and then gently rolled to ensure proper mixing of WCF with CIM (anticoagulant). An additional 500 L of WCF was collected from two of the 12 urchins assayed and transferred into microcentrifuge tubes; these aliquots of WCF/CIM were used as controls for the assay.
42 T o streamline the assay process, two trays were configured to hold 28 microcentrifuge tubes (one assay tray per experimenter). Rows were labeled with alphanumeric codes previously assigned to each urchin (C1, C2, and J U) and columns were labeled with the end times for each assay tube: 0mins, 30mins, 60mins, and 120mins. The intersection between the labeled row and column corresponded to the specimen and the assay length. This method allowed for mul tiple urchins to be assayed simultaneously. One hundred microliters of esoin Y stained yeast (at a concentration of 2,016yeast/mL FSW) was pipetted into all experimental assay tubes. Tubes that corresponded to the control groups were left empty. Aliquots of 200 L WCF/CIM were then added all assay tubes (both experimental and controls) to achieve a final ratio of 10:1 yeast cells per phagocyte in experimental assay tubes. The resulting yeast and phagocyte mixture was gently resuspended, each tube capped, the assa y tray carried to the lab hood, and left to incubate for 15 minutes. Immediately following incubation, 50 L of 4% formaldehyde was added to both experimental and control assay tubes at timed intervals: 0mins, 30mins, 60mins, and 120mins. The resulting f ormaldehyde and cell mixture was gently resuspended, and each assay tube was immediately capped to minimize the spread of formaldehyde vapors. Following fixation with formaldehyde, 10 L of the resulting WCF/CIM/yeast solution was added to sterile slide s, and smeared. One smear was prepared for each experimental and control group. Smears were allowed to air dry, then fixed for 30sec in 100% MeOH, and stained according to the methods described for the
43 coelomocytes of Apostichopus japonicas (C hen et al. 2008) To stain the smears approximately 1mL of PROTOCOL Wright Giemsa stain was added over each slide, allowed to sit for 2mins, and 1mL of FSW (pH 6.8) was added to the stain. The slides were then gently rocked for 1min to mix the FSW and Wright Giemsa and left to sit for an additional minute. Excess stain was drained, slides were gently rinsed with DI H 2 O, and finally blotted dry with Bibulous paper. Figure 13 : Flow Chart for Experiment 2: In Vitro Phagocytosis Assay (Steps 1 4). Steps 5 10 are detailed in Figure 14 A flow chart illustrating the experimental design of in vitro phagocytosis assays without tissues. 1. Urchins were first observed in individual holding tanks and their activ ities recorded (with respect to tube feet activity, spine activity, and crawling). 2. Yeast were then sterile assay tubes. 3. Five hundred microliters of WCF was then removed from each of 12 urchins and then gently rolled to ensure thorough mixing of WCF with CIM. 4. The color intensity of the WCF/CIM mixture was t hen recorded using a scale from 1(lightest) to 3 (darkest).
44 Figure 14 : Flow Chart for Experiment 2: In Vitro Phagocytosis Assay (Steps 5 10 ) A flow chart (continued from Figure 13 ) illustrating the experimental design of in vitro phagocytosis assays without tissues 5. Two hundred microliter aliquots of WCF/CIM were then transferred into 4 assay 120mins) for each of the 12 urchins assayed. 6. The yeast WCF/CIM mixture was then resuspended and incubated for 15minutes. 7. At timed intervals of 0 mins, 30mins, 60mins, and 120mins, 50 L of 4% formaldehyde was added to one of the four assay tubes prepared for each of the 12 urchins assayed. 8. Individual smears were made using the WCF/CIM mixture prepared from each urchin at each time d interval 9 Smears were allowed to air dry and were then stai ned using 10% Wrights Then, the cells on each smear were counted and the total phagocyte population ( TP ) and phagocytosis index (PI) determined. b. Experiment 3: Assays using E o sin Y stained Y east and U rchin T issue Eight urchins were assayed for their ability to eliminate eosin Y stained yeast from assay tubes containing coelomic fluid and freshly harvested urchin tissue in vitro. Two urchins were assayed for each of the followi ng tissues: gonads, digestive tract, peristomial gills, and peristomial membrane. T he sea urchin body plan differs from the worm: the urchin possessing an inflexible test with little to no connective tissue lining the inner cavity of the
45 peristomial coe lom and the worm having a flexible body wall lined with ample musculature (Figure 9 ) H arvest of musculature and body wall from the urchin was not feasible G onads, peristomial gills, digestive tract, and peristomial membrane were harves ted from the urchin and their e ffe ct on phagocytosis investigated Each o rgan was prepared separately and then assayed individually to examine the effect of each on phagocytosis in vitro An in vitro assay was completed similarly to the procedure described above however, for this assay freshly harvested tissue samples from the urchin gonads, peristomial gills, digestive tract, and peristomial membrane were placed in assay tubes. To examine the affect of tissue on L. variegatus phagocytes, tissues were harvested, and used in phagocytosis assays with sterile yeast as target cells. Urchin tissue was harvested, washed, and weighed ( Appendix 2) Two urchins were kept as controls for phagocytosis in the absence of tissue. A total of 2g tissue was harvested from the gonads, pe ristomial gills, digestive tract, and peristomial membrane and divided between eight (experimental) assay tubes such that there were a total of two experimental groups for each organ. Once all experimental assay tubes contained 0.5g of tissue, 200 L aliquots of eosin Y stained yeast were added to each experimental and control tube, the assay tray was gently rocked for 1 minute, and the mixture left alone for 15minutes. Following the 15min tissue/yeast incubation, 200 L of WCF/CIM was added to exper imental and control assay tubes such that the final concentration of yeast to coelomocyte was 10 : 1, the assay tray was gently rocked for 1 min, and allowed to incubate for 15mins. Immediately following incubation, formaldehyde was added to assay wells (at times
46 corresponding to 0mins, 30mins, 60mins, and 120mins), and 10 L aliquots of the resulting solution were used to prepare two smears for both experimental and control groups. The slides were stained with Wright Giemsa as described by Chen and others ( 2008 ) and finally blotted dry with Bibulous paper. Figure 15 : In Vitro Phagocytosis Assays with Tissues Flow Chart (Steps 1 5) Step s 6 14 are detailed in Figure 16 A flow chart indicating a simplified version of the experimental design of in vitro phagocytosis assays with L. variegatus tissues. 1. Urchins were first observed in individual holding tanks and their activities recorded (with respect to tube feet activity, spine activity, and crawling). 2. Yeast were aliquots were transferred into sterile assay tubes and set aside. 3. Gonads, digestive tract, peristomial gills, and peristomial membrane were then harvested over a bed of ice. 4. The or gans were then washed by three rounds of centrifugation (Appendix 2) and weighed into 0.5g sections. 5. Five hundred microliters of WCF was then removed from each urchin 1mL syringes (fitted with a 20 M, and then gently rolled to ensure thorough mixing of WCF with CIM.
47 Figure 16 : In Vitro Phagocytosis Assays with Tissues Flow Chart (Steps 6 14) A flow chart (continued from Figure 15) indicating a simplified version of the experimental design of in vitro phagocytosis assays with L. variegatus tissues. 6. The color intensity of the WCF/CIM mixture was then recorded using a scale from 1(lightest) to 3 (darkest). 7. The organs (weighed in 0.5g portions) were then transferred to the assay tubes containing yeast suspensions. 8. The organ/yeast mixture was resuspended and allowed 15 minutes to incubate. 9. Following the organ/yeast incubation, was then resuspended and allowed to incubate for 15 minutes. 11. At timed intervals of 0mins, 30mins, 60mins, and 120mins, 50 L of 4% formaldehyde was added to each assay tube. 12. Individual smears were made using the WCF/CIM mixture prepared from each urchin at each timed interval. 13. Smears were allowed to air dry and were then stained using 10% Wrights Then, the cells on each smear were counted and the total phagocyte population (TP) and phagocytosis index (PI) determined.
48 C. Analysis of P hagocyte A ctivity from S tained S mears To assess phagocytosis across different experimental conditions, cells were counted from stained smears that were representative of cellular activity from each experiment. Smears were stained with eit in vivo assay) or Wright Giemsa ( in vitro assays) stain. Phagocytes and yeast could both be visualized at a magnification of 20X using light microscopy. Smears that contained a large amount of cellular debris, or residual artifact from staini ng, were first observed at 20X, and then more closely inspected at 40X to obtain accurate cell counts. i. A C oordinate G rid for C ell C ounts Due to the large number of cells present on a single slide and the magnification needed to visualize them, it was n ot practical to obtain cellular counts from the entire slide. It was therefore necessary to devise an unbiased method to systematically perform cell counts. A grid system was designed by relating the dimensions of a standard frosted slide (= (75mm froste d portion) x 25mm) to the increments of a stage micrometer (Figure 1 7 ). Figure 17 : Diagram of Grid Construct Used for Cell Counts. Method used for cell cou nts. Figure 1 7 a. (modified from e olsurplus.com/Microscopes.html) F in e adjustment knobs (indicated) were used to move to coordinate points within a slide. Figure 1 7 b. Dimensions of the grid coordinate system. Values from the domain and range of the grid corresponded to the labeled increments of the stage micrometer. The f ro sted portion of slides was excluded from the grid and cellular counts.
49 The slide grid system was constructed around, and all cell counts were performed on, a single light microscope. Using a single microscope with the grid construct eliminated the intr oduction of errors from variations in the calibration of different stage micrometers. A random number generator was used to compile sets of random coordinate points using the domain [105,153] and range [2,22] of the grid construct In this manner severa l random coordinate points were generated and assigned to each slide prepared from in vivo and in vitro assays. D. Equations The methods used to evaluate phagocytosis assays were obtained and modified from Silva and colleagues ( 2001 ) The e ffect of L. varie gatus tissues on phagocytes and inflammation was evaluated by analyzing changes in the median number of phagocytes ( T P ) and the phagocyto sis index (PI) of phagocytes over time. Multiple smear s were prepared from the same animal and different animals under the same experimental condition were treated as replicates. The T P w as representative of the total number of phagocytes counted from each coordinate point on a slide. T he PI was a weighted index that represented the capacity for phagocytes to consume yeas t. Phagocytes that were not phagocytosing yeast were assigned a PI value of 1. (1 )
50 Chapter 3 Results This study served as a preliminary exploration of the phagocytotic coelomocytes from L. variegatus Th e morphologies, numbers, and activities of the coelomocytes within the whole coelomic fluid of L. variegatus were studied using light microscopy and are reported herein. The data obtained from experimentation are divided into two main sections; beginning w ith observations of the coelomocytes in hanging drops and wet mounts, and concluding with the results from the phagocytosis assays. I. Observations of the Coelomocytes Coelomocytes from L. variegatus were observed in wet mounts, hanging drops, and stained s mears from phagocytosis assays. Four types of coelomocytes were observed within the coelomic fluid of L. variegatus vibratile cells, red amoebocytes, colorless amoebocytes, and phagocytes. The proportion of each cell type within the coelomic fluid was not determined empirically; however it can be inferred that discoidal and small phagocytes were most common, red and colorless amoebocytes were less common and polygonal and vibratile cells were least common within the coelomic fluid. Three subpopulations of phagocytes were observed; polygonal phagocytes, discoidal phagocytes, and small phagocytes (Figure 1 8 a. ). The majority of phagocytes observed were discoidal and small phagocytes. Only small phagocytes wer e observed in the petaloid form ; the large phago cytes quickly adhered to the glass during viewings and transformed into the filopodial form. Discoidal and small
51 phagocytes were most often seen in cellular clots. Small phagocytes seemed to initiate the formation of syncytia over the glass slide. Observa tions indicate that small syncytia would initially form when a few small phagocytes would come in contact with other small phagocytes. Once in contact with other like cells, the small phagocytes would often make contact with the glass slide and spread onto it, transforming into the filopodial form, and forming a large mass of cells indistinguishable from one another. These syncytia were often observed to catch discoidal and polygonal phagocytes within them. The greatest number of discoidal phagocytes could be found within syncytia Once caught within syncytia, discoidal phagocytes would often find one another and begin to form cellular clots. The large polygonal phagocytes were also seen to adhere to one another when caught in sync y tia, although these cells were observed infrequently. Figure 18 : Images of Coelomocytes from Observations. Light microscope i mages of the coelomocytes from WCF at 2 0 0X in a wet mount. Figure 18 a. T wo phagocytes in the petaloid form Figure 1 8 b. An image of a phagocyte clot. Discoidal (*) and small phagocytes are seen forming this cellular clot. Discoidal phagocytes in the filopodial form (cells with long thin cellular projections), red amoebocytes, and a vibratile cell (blurred image indicated by a back arrow) can be all be seen within this cellular clot. 200X. Figure 1 8 c. A large polygonal phagocyte. 200X.
52 Cellular clots formed by discoidal and small phagocytes would often encompass red and colorless amoebocytes (Figure 1 8 b.) These clots were formed as colorless cytoplasmic projections, which were extended in all directions away from the cell body of discoidal phagocytes, became entangled with one another and small phagocytes. The number of these cytoplasmic extensions multiplied the longer the cells were observed until the cells formed an apparently singular mass. Vibratile cells were not observed to become caught within these cellular clots, but the cells were seen rapidly moving throughout the coelomic fluid, and adjacent to these cellular cl ots (Figure 1 8 b. ). The red amoebocytes were highly mobile and easily distinguishable from other cell types in preparations due to their intense red granules. The cells were observed to continually deform their cellular membrane. Cell blebbing was led by t he extension of a clear cytoplasmic protrusion, which would radiate about the cell body for several seconds to a minute before red granules were released into it, and another projection was extended. During the blebbing process, several adjacent granules w ithin the cell were observed to fuse with one another to form larger darkly colored granules (Figure 19 d e. ). This fusion of granules was most commonly observed during cellular pinocytosis, before internal degranulation. The red amoebocytes were also obs erved to (apparently) pinocytose particulate matter (Figure 19 ) This process was led by the extension of a cytoplasmic edge and followed by the distribution of red granules around the particle. Once surrounded by granules, the amoebocytes would further po sition the particle into the center of the cell, wherein one or more granules would internally
53 degranulate. The process of pinocytosis was rapid, occurring within minutes, and several amoebocytes could be seen participating in this process. Figure 19 : Images of A Red Amoebocyte from Observations I ntervals between images are not consistent. Figure 19 a c. Black arr ows indicate the extension, and subsequent filling of, a cellular e dge with red granules. Figure 19 d h. The b lue asterisk in dicates a cluster of granules (d .) that fuse to form a larger granule (e .), which is then moved into the cell body (f .) and then degranulates (g. & h .). Only discoidal phagocytes were observed to consume yeast from stained smears. A single discoidal phagocyte could consume from one to many yeast, a single small phagocyte was not large enough to consume a yeast cell on its own. Phagocytosis wa s most often observed when discoidal phagocytes and yeast cells had become localized within a net like network of small phagocytes. The formation of these net like masses of cells, or sync y tia of small phagocytes, was observed to effectively entangle and l ocalize most yeast from the coelomic fluid. II. Phagocytosis Assays The total number of phagocytes ( T P) and the phagocytosis index (PI) of experimental and control urchins were analyzed in order to determine if the presence of L variegatus tissues ( from dig estive tract, gonad, peristomial gills, and
54 peristomial membrane) cause d a significant change in the TP or PI of L. variegatus phagocytes in response to yeast. To evaluate the role of L. variegatus tissue in the process of inflammation, two central quest ions were asked and then examined in vivo and in vitro Firstly, does the presence of yeast cause an inflammatory response, as characterized by an increase in the total number of L. variegatus phagocytes (T P ) ? And secondly, does the presence of L. variegat us tissue cause a change in the efficiency of phagocyte yeast consumption (PI)? T o determine if L. variegatus tissues affected L. variegatus phagocytes two experiments ( in vivo and in vitro ) were conducted, and cells were counted from smears corresponding to them. It is important to note that cell counts from each organism could, and did, vary substantially from organism to organism. The fact that the variables examined in this study ( T P and PI) were calculated from the number of coelomocytes on smears, which could contain variable amounts of cellular content (such as cellular clots), and that coelomocytes were only counted from approximately 0.7 2.5% of the total slide area, indicate that each variable is sub ject to a high degree of error. Therefore, m edian scores were used for statistical analyses A. Statistics Medians were calculated using the number of cells counted at seven coordinate points from each slide. Slides prepared from assays with the same condition were treated as replicates (n = number of replicates). Replicates and medians were used to decrease the standard deviation of phagocyte counts. Using
55 points that could be used in statistical analyses. Therefore, st atistical te sts for significance were not used for all assay conditions. Non parametric tests for statistical significance were used to evaluate the median numbers obtained for the total number of phagocytes (TP) and phagocytosis indexes (PIs). A Mann Wh ittney test, which compares differences across two groups, was used to evaluate the variance between the numbers of phagocytes from assay conditions after 120 minutes in vivo Since sample sizes were small and variance unequally distributed, the Kruskal Wa llace test was used to evaluate the variance between the number of phagocytes from all assay conditions after 120 minutes in vitro A. Phagocytosis Assays: A Mann Whitney test for significance was used to analyze the differences between the median TPs found from FSW (n=4) and FSW+yeast (n=18) assay conditions after 120 minutes in vivo (Table 4 ) The differences between the TPs of these assay conditions and control conditions were not evaluated because there was only one median TP found. Only one median TP wa s found because out of the four slides prepared from the WCF of control urchins, two slides were stained too darkly and one slide had too many darkly stained aggregates to perform cell counts. Results from the Mann Whitney test showed that there was an alm ost significant (P = 0.0546) increase in the TP from FSW to FSW+yeast assay conditions in vivo (Table 4 ).
56 Table 4 : TP Mean and Standard Deviations After 120 Minutes In Vivo Condition: Control FSW FSW+Yeast Mean of Medians: 0.000 0.750 3.550 Sta ndard Deviation (SD): 0.000 0.500 3.745 Sample Size (n): 1 4 18 A Kruskal Wallis test for significance was used to analyze the differences between the median TPs found from FSW+yeast (n=15), FSW+yeast+digestive tract (n=4), FSW+yeast+gonads (n=4), FSW+ yeast+peristomial gills (n=4), and FSW+yeast+peristomial membrane (n=4) assay conditions after 120 minutes in vitr o (Table 5 and Table 6 ) It was found that there was a very significant (P = 0.0078) decrease in the TP from FSW+yeast to FSW+yeast+peristomia l gills assay conditions. Significant differences were not found between the TPs of other assay conditions in vitro Table 5: TP Mean and Standard Deviations After 120 minutes In Vitro Condition: No Tissue Digestive Tract Gonads Peristomial G ills Peristomial Membrane Mean of Medians: 7.433 5.250 1.000 0.625 4.500 Standard Deviation (SD): 4.280 1.983 1.732 0.479 1.732 Sample Size (N): 15 4 3 4 4 Note: all conditions listed above included FSW and yeast.
57 Table 6 : Comparisons of Median TP After 120 Minutes In Vitro Comparison: P value: No tissue vs. Digestive tract P > 0.05 No tissue vs. Gonad P > 0.05 No tissue vs. Peristomial gills P < 0.05 No tissue vs. Peristomial membrane P > 0.05 Digestive tract vs. Peris tomial membrane P > 0.05 Gonad vs. Peristomial gills P > 0.05 Gonad vs. Peristomial membrane P > 0.05 Peristomial gills vs. Peristomial membrane P > 0.05 Note: All in vitro assays contained FSW+Yeast in addition to the condition listed above. See Table 5 for the mean and standard deviation of median compared abovevalues. Indicate significant P values. There were no variances between the median PIs found from assay conditions in vitro or in vivo therefore a test for significance was not necessary.
58 C h a p ter 4 D iscussion I. Observations of the Coelomocytes Four types of coelomocytes were found within the whole coelomic fluid of L. variegatus vibratile cells, red amoebocytes, colorless amoebocytes, and phagocytes. Of the phag ocytes from L. variegatus discoidal, small, and polygonal subpopulations were observed (Figure 1 8 ) Discoidal and small phagocytes were most abundant within the whole coelomic fluid. Small phagocytes seemed to initiate the process of cellular clotting, ac ting as cellular nets that would entangle discoidal and polygonal phagocytes within them (Figure 1 8 b.) Polygonal phagocytes were not observed to adhere to discoidal or small phagocytes, but were observed to adhere to one another to form a large mass. Red amoebocytes cells were observed to pinocytose small matter and their granules observed to fuse with one another prior to internal degranulation (Figure 19 ) Observations of the small phagocytes in this study are in agreement with Brockton and others ( 200 8 ) that the small phagocytes initiate clotting. However, here I more specifically suggest that the phagocytes may initiate this process via a net like mechanism wherein the small phagocytes adhere to one another to form a network of small phagocytes that l ocalize coelomocytes within the coelomic fluid and promotes intracellular adhesion. These nets of small phagocytes would often encompass discoidal phagocytes, and the two would form a continuous mass of cells that would become indistinguishable from one an other over time. Although
59 polygonal cells could be observed in such networks of phagocytes and other cells, polygonal cells were not observed to form a continuous mass with discoidal or small phagocytes. Rather, the polygonal cells were only observed to ad here to one another. These large masses of polygonal cells were seen to encapsulate many yeast cells in vitro This suggest s that small phagocyte nets serve to augment encapsulation by catching many particles from the coelomic fluid (thereby concentrating their numbers in a localized mass) and localizing polygonal cell to this mass of particles in vivo The grouping of foreign entities into a concentrated mass would increase the rate and efficiency with which polygonal cells encapsulated them. II. Phagocytosis Assays After 120 minutes within the presence of yeast, statistical analysis revealed a nearly significant (P = 0.0546) increase in the number of phagocytes found in vitro ( Table 4 ) Therefore, the alternative hypothesis was supported The alternative hy pothesis s tate d th at if there wa s a significant change in the number of p hagocytes, then inflammation occurred. If it is assumed that the presence of FSW did not affect the number of phagocytes in vivo then the increase observed in the number of phagocyte s from FSW to FSW+yeast assay conditions can be solely attributed to the presence of yeast. Although not definitively backed by the test for statistical significance, the score obtained for the effect of yeast on the number of phagocytes in vivo indicates that the observed increase was due to the presence of yeast. This result was expected, the phagocytotic coelomocytes of echinoids are the primary line of defense against foreign intruders and the first to respond at sites of
60 host injury. It can therefore b e assumed that the phagocytes within the coelom of L. variegatus were able to recognize the injected yeast as foreign, respond to their presence by migrating from the connective tissues into the peristomial coelomic fluid, and therefore increase their numb ers. This finding is supported by many reports that y east initiates an inflammatory response mounted by t he phagocytotic coelomocytes within echinoderms and indicates that the methods used to evaluate changes in number of phagocytes in this study were succ essful (Adamowicz & Wojtaszek 2001, Bor ges et al. 2002, Tahseen 2008). S tatistical analysis also revealed that the number of phagocytes found in vitro very significant ly (P = 0.0078) decreased when in the presence of peristomial gill. No significant chan ges were found in the number of phagocytes between any other assays with or without tissues in vitro (Table 6 ) Therefore, the alternative hypothesis was supported and it was assumed that inflammation occurred In light of this data, it became necessary to develop a more complex hypothesis to accou nt for the phagocytes behavior. W hen the numbers of phagocytes were found to decrease, t he alternative hypothesis state d that the phagocytes were either forming brown bodies, cellular clots, and/or attempting to e ject themselves from the organism into the external environment. Based on this modified alternative hypothesis, i t was concluded that the number of phagocytes decreased from assays without tissue to assays with peristomial gills because they form ed brown b odies and cellular clots to trap and eliminate yeast. This result is supported by the earlier conclusion that phagocytes increased their numbers in response to the presence of yeast in vivo In this instance the phagocytes numbers did not increase because they
61 were not able to migrate out of the connective tissues into the peristomial coelomic fluid, but rather their numbers decreased as the phagocytes eliminated yeast by forming brown bodies and clots. If the formation of brown bodies and cellular clots were the only processes which accounted for the decreased number of phagocytes from assays without tissue to assays with peristomial gills, then there also would have been significant decreases observed from assays without tissue to all other assays with t issue (digestive tract, gonads, and peristomial membrane). However, there were no significant differences in the number of phagocytes between such assays. Therefore, there must have been another process responsible for the observed decrease in the number o f phagocytes from assays with peristomial gills. According to the alternative hypothesis, the only other explanation for this decrease wa s that the phagocytes were eliminated through excretory organs. However, it wa s not possible that the phagocytes number s decreased in vitro as result of excretion; it wa s highly possible that the phagocytes migrated into the peristomial gills. This result is supported by reports that phagocytes migrate into the respiratory trees and peristomial gills for excretion after ph agocytosing and encapsulating foreign invaders (Chia 1996; Johnson et al. 1970) If it is assumed that phagocytes migrated into the peristomial gills for excretion in vitro it can also be assu med that the phagocytes not only recognized the tissues but recognized them as viable tissue. If phagocytes were able to recognize peristomial gills then the phagocytes used in all in vitro assays may have
62 also been able to recognize tissue. Assuming that the phagocytes used within each assay were capable of recognizing tissues, it can be concluded that all tissues used in phagocytosis assays were viable since there were no significant differences between the numbers of phagocytes from assays without tissu e to all assays with tissue (excluding assays with peristomial gills). If it is true that all tissues used in phagocytosis assays were viable and that the phagocytes used within each assay were capable of recognizing them, then it can be concluded that the methods used in this study were a successful gauge for the effects of L. variegatus tissues on L. variegatus phagocytes. No significant results were obtained from the data relating to the phagocytosis ind ex of any of the groups analyzed There are many possible explanations for the lack of statistical evidence, but the most likely is that the sample size was too small. It was possible to generate statistically significant data from comparisons of phagocyte populations because there were a substantial nu mber data points for the number of phagocytes counted I n comparison to the total number of phagocytes counted from all smears there were only a small percentage of phagocytes consuming yeast. Therefore it was not possible to examine to effect of the vari ous experimental conditions on the phagocytosis index. III. Directions for F uture S tudies Observations of the coelomocytes in vitro have indicated that the small phagocytes may play a central role in localizing foreign invaders, phagocytes, and other coelomoc ytes within the coelomic fluid. These reports are consistent with
63 Brockton and others ( 2008 ) report that the small phagocytes may initiate clotting, and indicate that future studies should be directed toward ascertaining the role of the small phagocytes in clotting. The methods used for experimentation within this study were found to be effective for examining changes in the number of phagocytotic coelomocytes from L. variegatus in both the presence and absence of L. variegatus tissue. Phagocytes were obs erved to migrate into peristomial gills in vitro Since there was not enough data collected to analyze the statistical differences between assay conditions in vivo and in vitro there still remains a need for future studies to examine the e ffect of urchin tissues on the phagocytes. Before further research is conducted, the experimental design should be drastically modified. The greatest challenge of this study was data collection. Performing cellular counts was tedious, time consuming, and subject to a high degree of error since the phagocytes numbers were scored by the human eye. Therefore future studies should look toward modern techniques in order to minimize the degree of error implicit within cellular counting methods detailed in this study. It is rec ommended that future experimenters make use of a Boyden chamber, or a similar apparatus, when examining the coelomocytes with respect chemotaxis and phagocytosis. A Boyden chamber is composed of an upper and a lower chamber connected by a thin channel, wh ich allows for cellular movement between the two. This type of chamber can be easily used to evaluate chemotaxis in vitro ; cells are seeded in the upper chamber and the suspected chemoattractant in the lower. After a designated period of time the cells in both chambers are counted. If a significant
64 number of cells migrated into the chamber containing the suspected chemoattractant, it can be assumed to be chemotactic. Fisher Scientific distributes a modified 96 well Boyden chamber kit that allows for chemo taxis or phagocytosis to be quantitatively evaluated whilst eliminating the need to perform cellular counts. This assay kit comes with two opaque 96 well plates, which fit atop one another, and a fluorescently labeled membrane that is placed between the tw o. Cells are seeded into the upper plate, and chemoattractant into the lower (to replicate this experiment, combinations of FSW, yeast, and yeast with tissues would be placed the various wells the bottom plate). In order for cells to reach the wells of the bottom plate they must migrate through the fluorescent membrane. Transmigration through the labeled membrane transfers the fluorescent label to the cells. The number of cells that migrated through the membrane could then be easily determined using a stand ard 96 well plate reader. This method would not only simplify the experimental procedure described and implemented in this study, but would also allow for experimenters to rapidly generate data with a minimal degree of error. IV. Conclusions Observations of coelomocytes in vitro indicate that the small phagocyte mechanism, which localizes the phagocytes therefore increasing the rate at which they form clots. These preliminar ( 2008 ) report that small phagocytes may initiate clotting.
65 Statistical analysis indicated the presence of yeast probably induces inflammation in the tropical urchin Lytechinus variegatus This finding is in accordance with several reports of the phagocytes from many species of echinoderms ( Adamowicz & Wojtaszek 2001, Bor ges et al. 2002, Tahseen 2008). When in a solution of FSW, yeast, and tissue from peristomial gills, a significant number of phagocytes were found to migrate into the tissue in vitro This finding is consistent with reports from Chia and colleagues (1996) that the phagocytes migrate into respiratory trees and peristomial gills for secretion following particle encapsulation. Lytechinus variegat us tissue from digestive tract, gonad, and peristomial membrane was not found to significantly affect the number of phagocytes in vitro The role of these tissues on the phagocytosis index of the phagocyte population remains to be elucidated ; and there al so remains a need for further studies of the effects of L. variegatus tissues on the phagocyt osis index of phagocytes and the role of urchin tissue in the regulation of inflammation.
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73 Appendix 1 Solutions Recipes I. Filtered sea water (FSW): Sea water was pumped from the Sarasota Bay, filtered, sterilized, and kept on tap at the Pritzker Marine Laboratory of New College of Florida, Sarasota. To prepare filtered sea water (FSW), sea water from the Pritzker tap (pH 7.4; 29 0 / 00 ) was collected, passed through a 75mm Nalgene bottle top filter into a sterile container, and acidified to the physiological pH of urchin coelomic fl uid (pH 6.8 6.9) with concentrated HCl. II. Clot inhibiting medium (CIM): For 1L: 23.83g Hepes 21.04g NaCl 0.783g KCl 4.119g Na 2 SO 4 0.168g NaHCO 3 7.607g EGTA Combine dry ingredients in a 2L Erlynmier flask with a sterile stir bar. Bring solution to 1L with DI H 2 O and mix thoroughly. Store covered. III. Slide Cleaner: For 20mL: 20mL 70% EtOH 40 L 10M HCl Combine EtOH with 10M HCl and store covered under the laboratory hood.
74 Appendix 2 Instructions for Urchin Tissue Harvest and Preparation Samp les of L. variegatus tissue were collected from various organs and used in phagocytosis assays to determine if L. variegatus tissues affected the ability of L. variegatus phagocytes eliminated yeast. The methods of tissue harvest were modeled after previou s studies with L. terrestris (Marks et al. 1979) Due to the differences between the body plan of the urchin and worm, tissue was harvested from a number of urchin organs: digestive tract, peristomial gills, gonads, and peristomial membrane. The tissues were collected from multiple individuals and pooled to obtain a minimum of 2g from each organ. Each organ was assayed separately to analyze the effect of the different tissues on phagocyte function. I. Urchin dissection and tissue harvest: Several individuals were selected at random and dissected, using forceps and surgical scissors, on a dissection tray resting on a bed of ice. Phagocytes are induced to migrate into damaged and necrotic tissues, therefore it was of upmo st importance quickly harvest organs to minimize tissue necrosis and thoroughly wash the tissues to remove damaged cells. To begin the dissection, individuals were placed aboral side down, and an incision was made through the peristomial membrane and aro lantern, it was removed, and set aside. The coelomic fluids were drained and discarded, and the coelom was rinsed with FSW to remove debris. The urchin test
75 was then cut in half along a straight line from the oral to aboral side of the urchin using a pair of surgical scissors (Figure 2 0 a ). The inner cavities of the urchin were then inspected: urchins containing visible amounts of sperm (Figure 2 0 b. ) were not used for tissue harvest. Each organ was then removed in the following order: 1) gonads, 2) digestive tract, and 3) peristomial gills. Peristomial membrane was Care was take n to exclude spines, digestive material, and other organs from tissue preparations. Figure 20 : Diagram for Tissue Harvest from L. variegatus. Figure 20 a. The urchin oral side up with arrows represent the incision made from the oral to aboral al membran e (pm.) can be seen. Figure 20 b. The urch in split in two before washing. Figure 20 c. The urch in after washing. The top half of the urchin shows the urchin test after organs were harvested. The origin of the peristomial gills (pg.) is shown. Note that the gonads (g.) have released sperm (s.) therefore organ s from this urchin would not have be used. The digestive tract (dt.) and gonads (g.) are also shown above. Oral and aboral regions are indicated on the bottom half of the urchin. indicates organs harvested for assays.
76 II. Tissue washing and preparation: Following harvest, organs were carefully pic ked over for spines and debris using forceps, individually placed into sterile 10mL centrifuge tubes, and 10mL aliquots of FSW were added to suspend the tissues. Once suspended in FSW, the tissues were washed by three rounds of centrifugation at 600g for 5 minutes. The eosin Y Urchins that contained visible amounts of semen within the gonads were not used for organ harvest.