Investigations of celular dynamics during bleaching in the symbiotic anemone, Aiptasia pallida by Shanna D. Hanes A disertation submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degre of Doctor of Philosophy Auburn, Alabama August 3, 2013 Keywords: Coral bleaching, Heat stres, Aiptasia, Autophagy, Symbiodinium Copyright 2013 by Shanna D. Hanes Approved by Stephen Kempf, Chair, Asociate Profesor, Dept. of Biological Sciences Anthony Moss, Asociate Profesor, Dept. of Biological Sciences Scott Santos, Asociate Profesor, Dept. of Biological Sciences Richard C. Bird, Profesor, Dept. of Pathobiology, College of Vet. Medicine Bernhard Kaltenboeck, University Reader, Dept. of Pathobiology, College of Vet. Medicine ii Abstract The global decline of coral refs continues at an increasing rate despite eforts to identify key celular interactions responsible for the breakdown of these esential ecosystems. Coral bleaching involves the loss of esential, photosynthetic dinoflagelates (Symbiodinium) from host gastrodermal cels in response to temperature and/or light stres conditions. Although numerous potential celular bleaching mechanisms have been proposed, few studies have investigated the early host stres response when symbiotic breakdown is initiated. In this investigation, both celular and molecular techniques were employed in order to 1) carefully examine and characterize host anthozoan tisues at multiple stages of the symbiosis, including: i) healthy symbiotic, i) several stages of active heat stres induced-bleaching, ii) aposymbiotic (symbiont- fre); 2) document and describe al celular bleaching mechanisms that occur during heat stres treatment; 3) quantify any observed celular bleaching mechanisms in both symbiotic and aposymbiotic anemones to determine whether the response is host derived; 4) conduct a gene expresion analysis on heat stresed symbiotic A. pallida anemones in order to quantify the response using RNA-Seq on an Ilumina platform. First, histological and ultrastructural examinations were conducted using light and transmision electron microscopy on healthy symbiotic anemeone tentacle tisues in order to establish baseline information. A detailed ultrastructural analysis was conducted of numerous celular regions and compared to previous cnidarian literature. This study provided an esential diagnostic analysis of normal healthy tisues that was used to ases the health condition of the anemones during the subsequent heat iii stres treatments. Bleaching was induced by exposing both symbiotic and/or aposymbiotic A. pallida anemones, to ~32.5 o C at 120 ?mols iradiance for 12 h followed by 12 h in the dark at 24 o C daily for 2 days. Samples were taken thoughout the 48 h period. Ultrastructural examination revealed numerous autophagic structures and asociated celular degradation in tentacle tisues after ~12 h of stres treatment and also after 12 h of exposure to the known autophagy inducer, rapamycin. Additionaly, symbionts were observed detaching from highly degraded gastrodermal cels in an apocrine-like manner. The abundance of autophagic structures was quantified in gastrodermal and epidermal tisues of symbiotic and aposymbiotic tisues, and also in rapamycin treated tisues using ImagePro Plus 7.0 software. Results from the RNA-Seq analysis revealed the highest levels of diferential gene expresion in symbiotic A. pallida anemones after 3 h during a 48 h thermal stres treatment, suggesting that the gene expresion profile changes in early stages of the host stres response. In addition, several key proceses were identified that are involved in the host response, including stres response, protein degradation/synthesis, calcium homeostasis and others, which provides a beter understanding of the genetic determinants of stres tolerance in a host anthozoan. This investigation provided the first ultrastructural evidence of host autophagic degradation during thermal stres in a cnidarian system and supports earlier suggestions that autophagy is an active celular mechanism during early stages of bleaching. iv Acknowledgements I would like to thank my advisor, Dr. Stephen Kempf for the knowledge that he has bestowed on me over these years (I owe you a jerk mahi sandwich with mango chutney) as wel as my entire commite for their contributions to my disertation as wel as their support. I would also like to thank my faithful lab members, Maria Mays and Ivey Elis Holt, who have both served as my alies and great friends throughout al our lab adventures- Kempf Lab Unite! Mostly I would like to thank my family, who has shown me so much love, support, and aceptance over these years for following the beat of my own drum (despite how far marine biology may take from home). Mom and Dad, you are the greatest parents anyone could ever hope for, and Adam, Shari, and Ema, you are my inspiration! And I also must acknowledge Spyros Mourtzinis, who has stuck by my side through the darkest of days and served as my source of strength when I needed it most. He also contributed significantly to the development of this disertation. Thank you so much for everything, ?? ?????! I would also like to thank Dr. Bil Fit at UGA for alowing us aces to collection our Aiptasia specimens and also several undergrads that have worked in the labs, especialy Kristina Looney. v Table of Contents Abstract...................................................................................................................................ii Acknowledgments...................................................................................................................iv List of Tables.........................................................................................................................vii List of Figures.......................................................................................................................vii List of Abbreviations...............................................................................................................x I. Coral bleaching: Acelular breakdown in symbiosis..............................................................1 I. Histological and ultrastructural analysis of cel types within the tentacle tisues of the symbiotic anemone, Aiptasia pallida.......................................................................................19 Abstract ........................................................................................................................19 Introduction ..................................................................................................................20 Materials and Methods .................................................................................................22 Results and Discussion .................................................................................................24 Conclusions ..................................................................................................................51 II. Host autophagic degradation and asociated symbiont loss occur in response to heat stres in the symbiotic anemone, Aiptasia pallida.................................................................................54 Abstract ........................................................................................................................54 Introduction ..................................................................................................................55 Materials and Methods .................................................................................................58 Results and Discussion .................................................................................................63 Conclusions ..................................................................................................................73 vi IV. Autophagic activity occurs as a host-derived mechanism in the symbiotic anemone Aiptasia pallida during bleaching..........................................................................................................79 Abstract ........................................................................................................................79 Introduction ..................................................................................................................80 Materials and Methods .................................................................................................82 Results and Discussion .................................................................................................89 Conclusions ..................................................................................................................97 V. Diferential gene expresion responses to elevated temperature in symbiotic Aiptasia pallida anemones using RNA-Seq..........................................................................101 Abstract ......................................................................................................................101 Introduction ................................................................................................................102 Materials and Methods ...............................................................................................104 Results and Discussion ...............................................................................................108 Conclusions ................................................................................................................121 Literature Cited.....................................................................................................................123 General Conclusions.............................................................................................................142 vii List of Tables Table 1. Diferentialy expresed genes after 3 h and 48 h of thermal stres relative to 0 h controls categorized into functional groups.............................................................113 vii List of Figures Figure 1. Morphology of Aiptasia pallida and histology of symbiotic tentacle tisues.............25 Figure 2. TEM: Transverse sections of symbiotic Aiptasia pallida tentacle tisues..................26 Figure 3. TEM: Transverse sections of symbiotic gastrodermal tentacle tisues of A. pallida..28 Figure 4. TEM: Transverse sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues with gland cels containing various types of vesicular inclusions..............................31 Figure 5. TEM: Transverse sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues with gland cels containing putative zymogenic granules..........................................32 Figure 6. TEM: Transverse sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues containing putatative structures asociated with the gastrodermal nerve plexus.........34 Figure 7. TEM: Transverse sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues displaying a nutritive-muscular cel..........................................................................37 Figure 8. TEM: Transverse and longitudinal sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues at the region of atachment to the mesoglea.......................................39 Figure 9. TEM: Transverse sections of symbiotic gastrodermal Aiptasia pallida tentacle tisues at the region of atachment to the mesoglea...............................................................41 Figure 10. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues44 Figure 11. TEM: Transverse and longitudinal sections of symbiotic epidermal Aiptasia pallida tentacle tisues displaying longitudinal muscle cels.................................................45 Figure 12. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues displaying various cnidae..........................................................................................47 Figure 13. TEM: Transverse sections of symbiotic epidermal (A) Aiptasia pallida tentacle tisues displaying a sensory cel and asociated structures....................................................49 Figure 14. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues displaying a bacterial cyst.........................................................................................51 ix Figure 15. TEM: Transverse sections of control and unstresed treatment (t=0) symbiotic Aiptasia pallida tisues.............................................................................................64 Figure 16. TEM: Transverse sections of symbiotic Aiptasia pallida tisues after 48 h of exposure to heat stres.............................................................................................................6 Figure 17. TEM: Transverse sections of symbiotic Aiptasia pallida tisues after 48 h heat stres showing various stages of APS formation.................................................................67 Figure 18. TEM: Transverse sections of tentacles at midlength showing autophagic structure formation in the gastrodermis of symbiotic anemones...............................................69 Figure 19. TEM: Transverse sections of symbiotic Aiptasia pallida after ? 12 h heat stres showing apparent apical detachment of symbiont containing blebs from autophagicaly degraded host gastrodermal cels..............................................................................71 Figure 20. Average number of expeled Symbiodinium cels per anemone after 48 h of either heat stres or control treatment. (t-test, p < 0.05, n=3)......................................................72 Figure 21. Stages of autophagy...............................................................................................74 Figure 22. Quantification of autophagic structures within A. pallida tentacle tisues...............87 Figure 23. TEM: Transverse sections of symbiotic Aiptasia pallida tentacles at mid-tentacle length?....................................................................................................................91 Figure 24. Percent autophagic structures within the gastrodermis and epidermis of symbiotic control anemones and within the gastrodermis and epidermis of treatment anemones after 0 and 48 h of control treatment or heat stres....................................................92 Figure 25. TEM: Transverse sections of Aiptasia pallida tentacles at midlength......................94 Figure 26. Percent autophagic structures within the gastrodermis and epidermis of aposymbiotic control and treatment anemones after 0 and 48 h.......................................................95 Figure 27. Percent autophagic structures within the gastrodermis and epidermis of symbiotic control (12 h in 1% DMSO) and treatment (12 h in 25 ?M rapamycin + 1% DMSO) anemones..................................................................................................................96 Figure 28. Cluster analysis of diferentialy expresed genes from control anemones and anemones exposed to 3 h of thermal stres treatment..............................................110 Figure 29. Cluster analysis of diferentialy expresed genes from control anemones and anemones exposed to 48 h of thermal stres treatment............................................111 x List of Abbreviations APS autophagic structure Gv gastrovascular cavity ROS reactive oxygen species 1 I. Coral bleaching: A celular breakdown in symbiosis Coral-dinoflagelate symbiosis Coral refs are highly diverse and productive ecosystems that provide sustenance and shelter to a multitude of ref organisms, as wel as a wide variety of services to milions of people worldwide (NOA, 2012). Hermatypic (ref building) corals precipitate a calcium carbonate skeleton, which contributes over time to the formation of coral refs. Coral refs can support more species per unit area than any other marine environment (NOA, 2012). It is generaly acepted that the productivity of refs can be largely atributed to the establishment of close asociations or ?symbioses? betwen coral hosts and photosynthetic dinoflagelates of the genus Symbiodinium (Freudenthal, 1962), which are often refered to as ?zooxanthelae? (Hoegh-Guldberg, 1999). Most symbioses betwen dinoflagelates and their cnidarian hosts are obligate mutualisms that may have been selected for as a means to supplement nutrients in the oligotrophic tropical waters where most hermatypic corals are located (Muscatine and Porter, 1977). The coral-dinoflagelate symbiosis has been a subject of interest since each symbiotic member was individualy clasified in the late 19 th century (Brandt, 1881). Members of Symbiodinium spp. have been divided into 11 species that comprise at least eight groupings or ?clades? (A-H). The clades, in turn, contain multiple subclade types (Stat et al., 2006) that can be identified at the molecular level. Members of at least five invertebrate phyla are known to live symbioticaly with Symbiodinium spp. These include Protista, Porifera, Cnidaria, 2 Platyhelminthes and Mollusca (Hoegh-Guldberg, 1999; Stat, 2006). The variety of hosts harboring these symbionts is greatest in Cnidarians with many examples, such as hermatypic corals, found in the clas Anthozoa, subclas Hexacoralia (=Zoantharia). Such hosts maintain symbiotic relationships with a variety of Symbiodinium types (Weis et al., 2008; Sunagawa et al., 2009; Lenhart et al., 2012). Symbionts can be gained through two basic methods of acquisition by their cnidarian hosts, which include taking them up from the surrounding environment through horizontal transfer or gaining them directly from their parent through vertical transmision (Stat et al., 2006). Symbiont maintenance and nutrient exchange At the tisue level, the cnidarian bauplan consists of two celular layers, the external epidermis and internal gastrodermis, lying on either side of an acelular collagenous mesoglea. Symbionts reside intracelularly within nutritive-muscular cels of the gastrodermis, each present within a specialized host vacuole caled the ?symbiosome? (Wakefield and Kempf, 2001). The symbiosome membrane and the underlying symbiont secretions serve as the interface betwen host and symbiont, where esential nutrients are transfered betwen both members of the asociation. While harbored within host tisues, symbionts are sheltered from the external environment and receive host inorganic metabolic products, including amonia, phosphates, and CO 2 (Trench, 1979). In turn, symbionts may transfer as much as 95% of their photosynthetic products, including sugars, amino acids, and carbohydrates to their coral hosts (Trench, 1979). These esential nutrients provide the coral with the necesary energy for growth and calcium carbonate deposition (Muscatine, 1990), which contributes greatly to the fitnes of the coral (D'Elia and Wiebe, 1990; Muscatine, 1990). Any factor that reduces the eficiency of this 3 relationship wil have a major efect on the ecological succes of these obligate members of the symbiosis (Glynn, 1996). Bleaching response Throughout time, corals have adapted to survive despite a variety of naturaly occurring disturbances, including fluctuations in salinity (Coles and Jokiel, 1992), sedimentation (Rogers, 1990), and predation (Porter, 1972). However, the past several decades have produced a surge of anthropogenic impacts that have introduced new chalenges for corals and that threaten their future survival (Wilkinson, 1993; Donner et al., 2007). The danger for coral refs is complex. Although corals have historicaly demonstrated an ability to recover from natural disturbances, the anthropogenic-driven increase in frequency and severity of environmental stresors have contributed to the loss of over 19% loss of coral refs globaly in recent years (Wilkinson, 2008). Although a wide variety of environmental stresors currently threaten corals, including disease outbreaks (Kushmaro et al., 1997; Harvel et al., 1999) and ocean acidification (Hoegh- Guldberg et al., 2007), one of the major factors currently responsible for large-scale coral ref mortality is elevated sea temperature and/or high solar iradiance (particularly UV wavelengths) (Glynn, 1996; Hughes, 2003; Hoegh-Guldberg et al., 2007). When the thermal threshold of the host, symbiont, or both is exceded, the symbiont cels and/or asociated symbiont photosynthetic pigments can be lost from host gastrodermal cels (Hoegh-Guldberg, 1999; Fit et al., 2001). This proces, known as ?bleaching?, results in a lightening of the host coral as the underlying white calcareous skeleton is exposed through translucent coral tisue. Although corals can survive for a short period of time without their symbiotic partners, longer periods of time result in reduced reproductive capability (Szmant and Gasman, 1990), decreased growth and 4 calcification rates (Glynn, 1993), and death of the coral host (Brown, 1997; Hoegh-Guldberg, 1999). Breakdown of the symbiosis Collapse of the coral-dinoflagelate symbiosis is tightly regulated by stres responses of both members of the symbiosis. However, many studies have identified the photosynthetic apparatus of the symbiont as the first structure to be afected by temperature or light stres conditions (Leser et al., 1990; Iglesias-Prieto et al., 1992; Warner et al., 1999). During elevated temperature conditions, symbionts absorb more light energy than can be utilized for photosynthesis, thus reducing the available excitation energy for the electron transport chain (Smith et al., 2005). These events set in motion a state known as ?photoinhibition?, which involves photodamage to the photosynthetic apparatus of the symbiont (Warner et al., 1999; Leser, 2006). The damage is primarily targeted to the D1 protein located within the PSI reaction center (Warner et al., 1999; Smith et al., 2005). When the extent of D1 damage exceds the rate of repair a loss of functional PSI reaction centers occurs, photosynthesis declines (Smith et al., 2005) and the production of damaging reactive oxygen species (ROS) occurs (Franklin et al., 2004; Leser, 1996). Alternatively, symbiotic breakdown has also been shown to originate in the host as a result of mitochondrial membrane damage (Dykens et al., 1992; Ni and Muscatine, 1997; Dunn et al., 2012). The celular mechanisms involved with the initial heat and/or light stres response have not been as wel characterized in host cels as they have in symbionts. However, as is the case for chloroplast thylakoid membranes, damage to mitochondrial membranes can also produce high levels of ROS that wil inevitably difuse into host tisues (Dykens et al., 1992; Ni 5 and Muscatine, 1997; Dunn et al., 2012). If the rate of ROS generation exceds the rate of ROS detoxification, then acumulation of ROS may result in oxidative damage to celular structure (Leser and Farel, 2004; Richier et al., 2005), loss of cel function, and ultimately death of the afected cel (Chen and Gibson, 2008; Leser, 1997; Perez and Weis, 2006; Sherz-Shouval et al., 2007). Recent investigations suggest that such ROS-mediated stres is the underlying cause of symbiont loss during bleaching (Franklin et al., 2004; Leser, 2007; Perez and Weis, 2006). Reactive Oxygen Species Reactive oxygen species (ROS) such as superoxide (O 2 .- ), singlet oxygen (O 2 - ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( . OH), nitric oxide (NO), and peroxynitrite (ONO-) are an inevitable by-product of photosynthesis due to the diference in redox potential betwen the products and reactants of photosynthetic pathways (Niyogi, 1999). These molecules can directly damage lysosomal membranes, which serve a vital function by isolating potent degradative enzymes from other intracelular components (Kifin et al., 2006). ROS are normaly detoxified by antioxidants, such as catalase, superoxide dismutase and ascorbate peroxidase; however, when ROS production exceds the detoxification capability of a cel, membrane damage often results in leakage of potent lysosomal hydrolases that can significantly disrupt intracelular organization (Kifin et al., 2006) and lead to cel death. Mechanisms of Symbiont loss As a response to the various stimuli that trigger the bleaching proces, symbiont loss can occur via a number of potential celular mechanisms. These have been covered in several reviews (Gates et al., 1992; Douglas, 2003; Leser, 2004; Weis, 2008). 6 Exocytosis The first celular bleaching mechanisms suggested to result in loss of symbionts from host tisues was exocytosis. This proces has been implicated as a mechanism of bleaching in several studies (Yonge and Nichols, 1931; Sten and Muscatine, 1987; Brown et al., 1995). Sten and Muscatine (1987) exposed Aiptasia pulchela anemones to a low temperature of 4 o C, which induced bleaching after 8 hours. Scanning electron microscopy revealed that symbionts migrated to the apical ends of host gastrodermal cels, causing them to bulge into the gastrovascular cavity into which they were eventualy exocytosed. The released symbionts were expeled from the gastrovascular cavity in various stages of disintegration. They concluded that low temperature either increased the rate of microtubule depolymerization or increased cytosolic Ca 2+ , both of which could potentialy induce exocytosis. Fang et al. (1997) found that intracelular calcium steadily increased in coral cels undergoing hyperthermic stres. A subsequent study by Fang et al. (1998), also suggested that coral bleaching requires motor proteins in order to transport symbiont-containing vesicles via the cytoskeletal track to the cel membrane. Budding/Pinching off Glider (1983) first described this celular mechanism in his doctoral disertation while examining transmision electron micrographs of healthy Aiptasia pallida anemone tisues. The micrographs suggested that during this proces, initialy termed ?budding,? the cel membrane of host gastrodermal cels pinched inward, and symbionts along with surrounding host cel membrane and cytoplasm were released into the GVC. Observations of later stages revealed that 7 expeled symbionts were photosyntheticaly intact after release. The author hypothesized that this celular mechanism was a method of symbiont population control in non-stresed members of A. pallida. Gates et al (1992) later renamed this mechanism as ?pinching off? and suggested that it could be a potential celular bleaching mechanism. Host cel detachment This mechanism whereby bleaching occurred when gastrodermal cels containing intact symbionts were released from the mesoglea and expeled into the GVC, was initialy proposed by Gates et al. (1992). This response was observed after exposing the anemone Aiptasia pulchela and the coral Pocilopora damicornis to extreme temperatures of either 12 o C or 32 o C (10 o C above and below ambient temperature), both of which were beyond the temperature extremes recorded in the natural habitat from which these species were collected. Thus, it has been argued that this bleaching method only occurs as an extreme stres response, which is often followed by death of the host (Brown et al., 1995). The authors proposed that host cel release may occur as a result of cel adhesion dysfunction and calcium ion influx, however eforts to test this hypothesis were unsuccesful (Sawyer and Muscatine, 2001). In situ degradation This proces was first described by Brown et al. (1995) in corals that were collected from a bleaching event. Histological examinations of bleached tisues revealed numerous symbionts that appeared ?disrupted? and ?mishapen? within host gastrodermal cels. Degraded symbionts were observed being exocytosed from host cels. However, within tisues that had been subjected to an advanced state of thermal stres, host cels containing symbionts appeared to be released 8 from the gastrodermis (se host cel detachment). Symbionts showed a spectrum of morphologies from normal to completely degraded, which suggested that progresive degradation was occurring in situ prior to the actual loss of symbionts from host tisues. Several other reports of in situ degradation of symbionts have been described in both naturaly bleaching corals (Le Tisier and Brown, 1996; Ainsworth and Hoegh-Guldberg, 2008) and experimentaly heat stresed corals, as wel as in anemones (Dunn et al., 2004; Franklin et al., 2004; Strychar et al., 2004). However, most observations of in situ symbiont degradation were claimed to be the result of other degradative events, such as apoptosis or necrosis, rather than as an independent bleaching mechanism (Dunn et al., 2004; Franklin et al., 2004; Strychar et al., 2004; Ainsworth and Hoegh-Guldberg, 2008). There stil remains much uncertainty over whether the appearance of degraded symbionts in situ is a result of ROS-mediated cel death activity or if the proces functions as a host-controlled independent bleaching mechanism. Cel death mechanisms Cel death is an important function for proper development, controling cel numbers and eliminating abnormal or damaged cels or invasive pathogens (Kourtis and Tavernarakis, 2009). Thre major types of cel death have been defined, based on morphological criteria (Edinger and Thompson, 2004), apoptosis, autophagy, and necrosis, al of which have been implicated in cnidarian bleaching. Although these forms of cel death are morphologicaly independent proceses, there is growing evidence that some may be interelated (Xue et al.,1999; Klionsky and Emr, 2000; Dunn et al., 2002; 2004; 2007; Abraham and Shaham, 2004; Kourtis and Tavernarakis, 2009). 9 Apoptosis: Type I cel death Apoptosis is a type of programed cel death (PCD) that is a critical proces in cel homeostasis and the deletion of damaged or unwanted cels. In vertebrates, apoptosis is highly complex and involves multiple interacting pathways that eventualy lead to death of the cel. This proces is characterized by distinctive morphological features, including cel shrinkage, chromatin condensation, and nucleosomal DNA fragmentation (Edinger and Thompson, 2004). During the final stages of apoptosis, the entire cel is packaged into ?apoptotic bodies? which are ingested by phagocytes. The remnants of the cel are removed by phagocytic or neighboring cels (Gozuacik and Kimchi, 2004), thus no inflamation of surrounding tisues occurs (Edinger and Thompson, 2004). Genetic regulation of apoptosis Apoptosis is regulated by the activation of caspases, which are a family of cysteine proteases that undergo proteolysis and activation by other proteins and/or transcription factors (Earnshaw, 1999). Apoptotic activation can be induced by either the receptor-mediated, ?extrinsic? pathway or the mitochondrial, ?intrinsic? pathway (Lawen, 2003). The extrinsic pathway is executed when receptors such as tumor necrosis factor receptor (TNF-R), located on the plasma membrane of the cel, are activated by extracelular ligands (Lawen, 2003). This leads to the activation of initiator caspases (Caspase-8 or -10) that initiate executioner caspases (Caspase-3, -6, -7) that degrade celular targets (Lawen, 2003). Alternatively, intrinsic-mediated apoptosis is often activated by a variety of intracelular ROS, NOS, or cytotoxic stres/damage to the outer mitochondrial membrane (Brune et al., 1999). These stresors activate regulatory proteins, such as bcl-2, which are also embedded in the outer mitochondrial membrane. These 10 factors al initiate the release of pro-apoptotic molecules, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol (Lawen, 2003), where it binds to Apoptotic Protease Activating Factor (Apaf-1)/Caspase-9 complex that initiates executioner caspase, Caspase-3. Apoptosis during bleaching Caspases and other apoptosis genes are highly conserved among taxa and have been connected to the bleaching proces in cnidarians. It has been suggested that during bleaching apoptosis acts to mitigate tisue damage from ROS by eliminating damaged cels (Dunn et al., 2004). A similar proces occurs in higher taxa where apoptosis is utilized to remove invading microbes if they manage to evade host innate imune defenses (James and Gren, 2004). It?s also an esestial celular proces that plays an important in regulating developmental proceses. Autophagy Although primarily a homeostatic response, the presence of autophagic structures in dying cels has recently implicated autophagy as a Type I cel death proces (Kourtis and Tavernarakis, 2009). Autophagy is a proces of cel recycling through lysosomal degradation that is esential for cel survival, diferentiation, development, and homeostasis through cytoplasmic, protein, and organele turnover (Holtzman, 1989). The autophagic proces is generaly acepted as a ubiquitous activity in eukaryotic cels (Ohsumi and Mizushima, 2004), and both protective and destructive contributions have been reported, largely from morphological observations of cel activities. Using autophagy, cels dispose of obsolete, exces or damaged parts, such as mitochondria, peroxisomes and regions of the Golgi, but the proces can also result in cel death 11 (Cuervo, 2004). Autophagy is primarily characterized by the increased appearance of double membrane-bound cytoplasmic vesicles (autophagosomes) that engulf bulk cytoplasm and/or cytoplasmic organeles. These autophagic vesicles and their contents are then destroyed by the lysosomal system of the same cel. Stages of the autophagic degradative proces The proces of autophagic lysosomal degradation consists of several sequential steps involving sequestration, transport of lysosomes, degradation, and utilization of degradation products (Holtzman, 1989). First, various components of the cytoplasm, long-lived proteins, and intracelular organeles are sequestered within cytoplasmic double-membrane vesicles caled autophagosomes or autophagic vacuoles (Kourtis and Tavernarakis, 2009). Young autophagosomes arise as regions of cytoplasm surrounded by a cup-shaped ?pre-autophagosomal structure? that eventualy fuses with itself to seal of or ?sequester? the vacuole?s contents from the rest of the cytoplasm and form the actual ?autophagosome? (Holtzman, 1989). These structures are generaly termed ?autophagolysosomes? after they fuse with primary lysosomes, which contain acid hydrolases that degrade the sequestered materials (Fawcet, 1981; Jing and Tang, 1999). The apparent continued sequestration of cytoplasmic contents by autophago(lyso)somal structures prior to or after fusion with the primary lysosome has been observed in the anemone, Aiptasia pallida, during heat stres induced-autophagy (Chapter 3); however, whether this is a common occurrence in other cnidarians is unknown. Breakdown of sequestered materials by lysosomal hydrolases results in release of the digested materials back into the cytoplasm to be recycled for macromolecular synthesis and/or ATP generation (Wang and Klionsky, 2003). As digestion proceds, the autophagolysosomes eventualy condense into 12 electron dense, heterogeneous, indigestible materials within the autophagosome membrane (Fawcet 1981; Holtzman 1989). These end products of autophagy are caled residual bodies. Role of autophagy during stres or injury Although autophagy is regularly sen under normal, non-pathological circumstances in many cel types, it may be induced under conditions of stres such as hyperthermia, which often results in cel damage or death (Schwartz et al., 1992; Swanlund et al., 2008). However, autophagy has also been implicated as a pro-survival proces for its response to intracelular stres conditions, whereby fre amino and faty acids are produced as metabolic substrates for adaptation to stres. In the absence of autophagy, the turnover of cytoplasmic proteins is impaired, which increases the likelihood they wil become damaged, misfolded, ubiquitinated and removed (Hara et al., 2006; Komatsu et al., 2006). Defective autophagic activity contributes to a number of diseases, including myopathies, neurodegenerative diseases (e.g. Parkinson?s and Alzheimer?s), and some forms of cancers (Kelekar, 2006). Recent studies have indicated that ROS production induces autophagy (Sherz-Shouval and Elazar, 2007), which plays a role in eliminating proteins damaged during oxidative stres (Xiong et al., 2007). Co-involvement of autophagy and apoptosis Based on the fact that both autophagy and apoptosis share several key genes and transcription factors in their respective molecular pathways, there is growing evidence that the two proceses are interelated (Xue et al., 1999; Klionsky and Emr, 2000; Abraham and Shaham, 2004; Dunn et al., 2007; Kourtis and Tavernarakis, 2009). Several pro-apoptotic signals induce 13 autophagy, whereas signals that inhibit apoptosis also inhibit autophagy; however, the exact nature of the relationship betwen autophagy and apoptosis is complex and not fully understood (Kourtis and Tavernarakis, 2009). Since autophagic activity is often detectable in regions where apoptosis is occurring, it has been suggested that autophagy is a type of non-apoptotic PCD (Tsujimoto and Shimizu, 2005). Alternatively, autophagy may also precede apoptosis as a defense mechanism (Lockshin and Zakeri, 2004) or function to ensure cel death in the event of inhibition of other death pathways (Kosta et al., 2004). Autophagy during bleaching Recent investigations of cnidarian bleaching corroborate this idea by providing evidence that autophagy is interlinked with apoptosis during hyperthermic stres (Dunn et al., 2007). In A. pallida, Dunn et al. (2007) demonstrated that chemical induction of autophagy by rapamycin caused masive bleaching at ambient temperature (24 o C) suggesting that autophagy can play a role in symbiont regulation. However, elevated temperature-induced bleaching was represed only when both apoptosis and autophagy were inhibited simultaneously. Thus, it was hypothesized that the two forms of cel death were interconnected, such that when one is inhibited, the other is induced. A more recent investigation (se Chapter 3) documented autophagic activity in A. pallida tisues exposed to heat stres conditions. In this study, anemones treated with rapamycin, a known autophagy inducer (Noda and Ohsumi, 1998), exhibited the same ultrastructural characteristics as heat stresed tisues, confirming that the structures observed during heat stres treatment were autophagic. Rapamycin-induced (and heat- induced) autophagic structures contained sequestered host celular material and were often found within highly degraded regions of the cel that exhibited an abnormaly sparse cytoplasm. 14 In addition, a novel bleaching mechanism termed as ?apical detachment? was observed in the same thermaly stresed tisues undergoing active autophagy. This bleaching mechanism was characterized by a series of celular events that occurred in autophagic host cels. These steps involved the i) gradual movement of the healthy symbiont within its symbiosome toward the apical region of host cels i) bulging of symbiont/symbiosome with asociated host cel plasma membrane and cytoplasm into gastrovascular cavity, ii) detachment of apical portion of host cel along with healthy symbiont/symbiosome in an apocrine-like manner into the gastrovascular cavity. The authors suggested that heat stres induced autophagic degradation led to reduced celular stability, eventualy resulting in the loss of symbionts via this apical detachment mechanism. Symbiophagy: One recent study measured elevated autophagic activity in the heat stresed coral Pocilopora damicornis using markers of autophagy (Rab 7 and LAS) (Downs et al., 2009). Histological examinations of bleaching tisues revealed an increase in the vacuolar space betwen the symbiont and host tisues with length of thermal treatment. Downs et al. (2009) hypothesized that symbionts were digested by the host through an innate intracelular protective pathway termed ?symbiophagy?. This term was modified from the proces known as ?xenophagy?, which involves the digestion of potential intracelular pathogens. The authors suggested that during symbiophagy, the vacuolar membrane (or symbiosome) that envelopes the symbiont is transformed from a conduit of nutrient exchange to that of a phagolysosome that is then recognized by the lysosomal system. As a result lysosomes fuse with the former 15 symbiosome leading to the digestion of the symbiont. Experimental data supporting this hypothesis remains to be provided. Molecular studies of autophagy Autophagy was discovered in mamalian cels but has been extensively studied in yeast (Huang and Klionsky, 2002), where over 20 genes encoding proteins involved in autophagy (designated as AuTophaGy related - ATG) have been identified (Klionsky et al., 2003). The basic mechanism of autophagy has been wel conserved among taxa and al share a similar set of ATG genes (Tsujimoto and Shimizu, 2005). Autophagy is controlled by several proteins, including the PI3IK/Akt complex which regulates another major protein kinase, mamalian Target Of Rapamycin (mTOR), which negatively regulates the pathway (Kamada et al., 2000). After the induction of autophagy, several regulatory stages mediated by various proteins and/or transcription factors must occur in order to complete the proces. Downstream of mTOR kinase, membrane nucleation occurs by the joining of Beclin 1, a human homolog of the yeast autophagy gene ATG 6, the vacuolar protein sorting protein (VPS 15), and the kinase PI3K II. These subunits are regulated by the anti-apoptotic/anti-autophagic protein, Bcl-2, which also has a nuclear export signal (Kang et al., 2011). Numerous ATG proteins are esential for the later stages of autophagy (Levine and Klionsky, 2004), whereby celular contents are sequestered into autophagosomes, which then fuse with lysosomes and degrade celular contents. Cel Necrosis: Type II cel death Lastly, necrotic cel death is defined as non-lysosomal vesiculate degradation, which is characterized by the dilation of intracelular organeles and breakdown of the plasma membrane 16 that often causes inflamation of surrounding tisues (Edinger and Thompson, 2004). Cel necrosis is most often triggered by extrinsic factors, such as physical injury, that cause the cel and its organeles to swel and eventualy rupture (Wyllie et al., 1980). In contrast to apoptosis and autophagy, necrosis has been clasified as uncontrolled cel death; however, recent investigations suggest that programed cel necrosis may occur (Edinger and Thompson, 2004). Several genes have been proposed to regulate programed necrosis, such as the protein kinase RIP (receptor interacting protein) and poly (ADP-ribose) polymerase (PARP), which negatively regulate the caspase-independent pathway (Proskuryakov et al., 2003; Edinger and Thompson, 2004). Cel necrosis during bleaching Both cel necrosis and apoptosis were documented in host tisues and in the symbiont according to morphological appearance of cels in thermaly stresed A. pallida (Dunn et al., 2002; 2004). In these studies, Dunn and colleagues subjected anemones to a range of elevated temperatures for varying lengths of time. These studies revealed a shift from apoptosis at the lower stres levels, i.e. moderate temperature stres and shorter duration, to necrosis at the more severe stres levels. This led to the hypothesis that apoptosis acts to mitigate tisue damage from ROS at moderate stres levels, thereby maintaining tisue homeostasis by eliminating damaged cels. However, this control is lost under severe stres, where necrosis predominates. 17 Sumary of potential methods of symbiont loss Al the previously described celular bleaching methods (cel death and non-cel death- related) were observed in a variety of cnidarian species under a range of thermal stres conditions. Thus, there is stil no agrement on which method primarily occurs in nature, if any (Weis, 2008). The celular mechanisms involved in the cnidarian bleaching proces are a complex set of interactions betwen two symbiotic members that are afected by many environmental factors. Contrasting descriptions of bleaching (mechanisms of symbiont loss and relative contribution of zooxanthelae versus pigment loss) may reflect diferences betwen species, the nature of the stresor and/or the amount of time lapsed betwen the onset of bleaching and collection of samples (Brown et al., 1995; Chapter 3). While some of these experiments have been succesful in simulating ?natural? bleaching conditions in the field, others have used conditions that are not typicaly experienced during natural conditions. Thus, extrapolation of potential methods of bleaching to those recorded during natural bleaching conditions in the field should be conducted with caution (Brown et al., 1995; Chapter 3). To further complicate maters, the majority of evidence for the proposed methods largely comes from histological snapshots of bleaching tisues. Therefore, the celular dynamics underlying these events remain unresolved (Weis, 2008). 18 Conclusions The earliest reports of bleaching events occurred in the late 19 th century (Glynn, 1993). Over the past few decades, an increasing frequency of bleaching episodes has been documented (Gates et al., 1992, Hughes, 2003; Hoegh-Guldberg et al., 2007), and these have impacted refs on a more global scale (Hoegh-Guldberg, 1999). Recent evidence suggests that since 18,000 years ago, tropical oceans have fluctuated in temperature by les than 2 o C (Thunnel et al., 1994). Thus, the cnidarian-dinoflagelate symbiosis has evolved to remain stable within a narow temperature range. However, in the past 100 years sea surface temperatures have increased by almost 1 o C and are curently increasing at the rate of approximately 1-2 o C per century (Hoegh- Guldberg, 1999). Since corals are currently living close to their thermal maxima, even smal increases in temperature (1-2 o C) can disrupt their symbiosis with Symbiodinium (Glynn, 1990; Hoegh-Guldberg, 1999). As a result, corals wil continue to be threatened by increasing frequency and severity of bleaching events in combination with other anthropogenic-driven disturbances, such as coral disease and ocean acidification that often co-ocur (Brown, 1997). If alowed to continue unchecked, these environmental impacts wil have grave negative consequences on the health of ref ecosystems. 19 I. Histological and ultrastructural analysis of cel types within the tentacle tisues of the symbiotic anemone, Aiptasia pallida Abstract Coral bleaching involves the loss of esential, photosynthetic dinoflagelates (Symbiodinium sp.) from host gastrodermal cels and occurs as a stres response of both members of the symbiosis. Although this phenomenon has been thoroughly investigated for several decades, the complex set of celular interactions involved in the breakdown of the symbiosis are only beginning to be unveiled. Thus, a detailed ultrastructural analysis of a commonly used model symbiotic cnidarian is critical in order to provide esential baseline information for future bleaching investigations to ases celular changes in response to stres conditions. In this study, a detailed ultrastructural overview of the celular and sub-celular structures that underlie a healthy stable cnidarian-dinoflagelate symbiosis was caried out using the Aiptasia ?Symbiodinium system. Both light and transmision electron microscopy (TEM) were used to examine multiple regions throughout the tentacles of A. pallida. Ultrastructural investigations revealed various celular structures, some of which were similar in appearance to those previously described in other cnidarians while other structures appeared diferent from those in other cnidarians in the ultrastructural literature. Numerous cel proceses were observed penetrating the mesoglea, which serves as acelular substrate for the atachment of al epithelial cels. Longitudinal sectioning revealed finger-like projections of mesoglea extending betwen adjacent gastrodermal cels and cel proceses. These observations suggest that in A. pallida, the mesoglea is a complex matrix that extends amongst the entangled proceses of adjacent epithelial 20 cels rather than forming a simple atachment along the bases of these cels. This complex structure has direct implications for a suggested celular bleaching mechanism known as ?host cel detachment.? This study represents the first detailed and systematic analysis of the Aiptasia model system or any other symbiotic cnidarian. Futhermore, the ultrastructural information gained from this analysis can be used as an informative health asesment tool as they are manipulated in the lab or during field-mediated conditions. Introduction Coral refs serve as the trophic and structural foundation of the ref ecosystem, a tropical oasis that boasts the highest diversity of marine organisms on the planet (Hoegh-Guldberg, 1999; Stat et al., 2006). Corals and other cnidarians belonging to the clas Anthozoa, subclas Hexacoralia (= Zoantharia) are exclusively marine (Fautin and Romano, 1997). This group includes scleractinian corals as wel as sea anemones that form symbioses with photosynthetic dinoflagelates from the genus Symbiodinium (Freudenthal, 1962). The intracelular symbionts comprise at least eight clades (A-H), which contain multiple subclade types (Stat et al., 2006) that can be identified at the molecular level. Although hosts from five invertebrate phyla are curently known to harbor these intracelular symbionts, cnidarian-algal symbioses have gained the most atention in recent years (Stat et al., 2006). This primarily results from the rapid decline in coral abundance and diversity that has resulted from anthropogenic-linked environmental stresors (Hoegh-Guldberg et al., 1999, 2007). During stresful conditions, such as elevated temperature and light, the symbiotic relationship betwen the cnidarian host and the symbiotic cels breaks down through a series of 21 celular events known as ?bleaching?. Cnidarian bleaching has been extensively studied for the past few decades. However there is stil much uncertainty surrounding the celular events that are involved during a natural bleaching episode (Weis, 2008). Previous investigations have described a wide variety of celular bleaching mechanisms resulting from examinations of temperature or light stresed cnidarian tisues (Taylor, 1973; Glynn et al., 1985; Sten & Muscatine, 1987; Gates et al., 1992; Brown et al., 1995; Dunn et al., 2002; 2007; Franklin et al., 2004; Strychar et al., 2004; Richier et al., 2006; Downs et al., 2009; Chapter 3). Yet one of the major impediments to these studies was that many of them were based on histological or ultrastructural ?snapshots? of the stresed tisues rather than conducting a proper comparison to control unstresed tisues over the time course of the bleaching episode. Such a comparison is critical when conducting ultrastructural analyses of cnidarian tisues, which can be quite complex and dificult to interpret after experimental manipulations without a thorough understanding of control tisues beforehand (Hanes, personal observation). In addition, it has been suggested that the utilization of an experimentaly tractable model organism, such as the anemone symbiosis model, Aiptasia spp. (Order Actiniaria) would greatly aid future celular investigations (Weis et al., 2008). For example, Aiptasia spp. maintain symbioses with a variety of Symbiodinium spp. types in a manner similar to what is sen in many corals (Weis et al., 2008; Sunagawa et al., 2010; Lenhart et al., 2012). The absence of a calcareous skeleton in anemones also enables most celular and microscopical manipulations to be conducted with much les efort than if using scleractinian corals (Sunagawa et al., 2010; Lenhart et al., 2012). Thus, a detailed ultrastructural analysis of this commonly used model symbiotic cnidarian is critical in order to provide esential baseline information for future bleaching investigations to use to ases celular changes in response to stres conditions. 22 Numerous early studies provided a detailed look at the histology and ultrastructure of cnidarians using Hydra as their model (Hes et al., 1957; Slautterback and Fawcet, 1959; Wood, 1961; Gauthier, 1963; Slautterback, 1967; Davis and Haynes, 1968; Haynes et al., 1968; Haynes and Davis, 1969; Rose and Burnet, 1968; Weis, 1971; Haynes, 1973; Weber and Schmid, 1985). More recent investigations have aimed to describe the ultrastructure of a wide variety of anthozoans (Westfal et al., 1973, 1997, 1998; 2002a; Fautin and Mariscal, 1991; Westfal and Eliot, 2002), including A. palida (Westfal et al, 1997, 1998, 1999, 2001, 2002; Westfal and Eliot, 2002). However, most of the ultrastructural studies conducted by Westfal and colleagues have focused on identifying neural synapses with various cels types (Westfal et al, 1998, 1999, 2001) along with describing neural pathways (Westfal et al., 2002; Westfal and Eliot, 2002). Therefore, there are numerous aspects of the celular ultrastructure of A. pallida that have yet to be described. In this study, a detailed ultrastructural analysis of the tentacular tisues of healthy, unstresed Aiptasia pallida provide an overview of the celular and sub-celular structure that underlies a healthy cnidarian-dinoflagelate symbiosis. Materials and Methods Culture conditions Aiptasia pallida harboring symbionts typed as Clade A4 Symbiodinium (Santos et al. 2002; Scott Santos, pers. comm.) were collected in the Florida Keys and maintained in artificial seawater (Ref Crystals) at 28-30 ppt salinity. Anemones were kept in two 150 gal tanks maintained at an average temperature of 24?1?C (such notation indicates mean ? standard deviation). The bottom of each tank, where most anemones were located, was ~1 m below a light 23 source that covered the length of each tank. The light source consisted of two fluorescent light fixtures per tank that were each equipped with two 32W bulbs (Philips F32T8/TL841) producing a 50?4 ?mol photons m -2 s -1 iradiance at the bottoms of the tanks. Lights were set on a 12:12 h light/dark regime. The anemones were fed freshly hatched naupli of Artemia thre times per wek. Anemone preparation for light (LM) and transmision electron (TEM) microscopy Twelve medium sized anemones were placed in identical, individual dishes that each contained ~200 mL 0.45 ?m Milipore-filtered artificial aquarium sea water (MFAW) and were alowed to aclimate for ~5 d to the ambient lab temperature of 24?1 o C with 50?4 ?mol photons m -2 s -1 ambient light intensity. Water in the dishes was changed daily, and the anemones were fed once during the first 3 d. They were then held unfed for 48 h prior to excision and fixation of tisues. Anemones were al prepared for fixation by removing the seawater from each anemone container and replacing it with ~300 mL high Mg/low Ca seawater (Audesirk & Audesirk, 1980) for 30 min, followed by the addition of ~2 mL chloretone saturated seawater for an additional incubation time of 30 min. Next, a subset of tentacles was clipped from each of 6 randomly selected anemones. Tisues were fixed using methods similar to the protocol of Caroll & Kempf (1994) in a primary fixative solution of Milonig?s phosphate-buffered 2.5% glutaraldehyde containing 0.14M NaCl for 1 h, followed by rinses with a 1:1 mixture of 0.34M NaCl and Milonig?s phosphate buffer solution. The tentacles were secondarily fixed in a 1.25% NaHCO 3 buffered 2% OsO 4 solution for 1 h. Folowing secondary fixation, tentacles were rinsed thre times in 1.25% NaHCO 3 buffer , dehydrated through an ethanol series to 100% ethanol, 24 transfered through thre changes of propylene oxide, and infiltrated and embedded using EMbed 812 resin (Electron Microscopy Sciences). Embedded tentacles were sectioned at either 0.5 - 1 ?m or ~60 nm thicknes using a diamond knife (Diatome) on a Reichert-Jung Ultracut E microtome. Sections cut at 0.5 ? 1 ?m were stained with Richardson?s stain for LM. Sections cut at 60 nm for TEM were stained with uranyl acetate and lead citrate. Tisues were visualized and photographed in sections from mid-tentacle length and around the circumference of each tentacle using a Zeis EM 10C 10CR transmision electron microscope. Negatives from TEM were scanned as positives on an Epson Perfection 3200 Photo flatbed scanner at 1200 dpi, and contrast and levels were adjusted using Adobe Photoshop 8.0. Results and Discusion A. palida exists as a solitary polyp that ataches to the substrate at the basal disk, extends upward along a column that terminates as the oral disk surrounded by numerous radiating tentacles (Fig. 1A). As is the case with al anemones, the column and tentacles (Fig. 1 A & B) consist of an epidermal and gastrodermal tisue layer separated by an acelular collagenous layer caled the mesoglea (Fig. 1 C & D). The mouth at the center of the oral disk (Fig. 1B) opens into the gastrovascular cavity (Gv) that extends throughout the column and into the tentacles (Fig. 1C). A. palida and many other diploblastic cnidarians harbor their symbionts within the cels of the digestive tisue (gastrodermis) (Fig. 1C, D and 2). 25 Figure 1. Morphology of Aiptasia palida and histology of symbiotic tentacle tisues. A. From the side view, the anemone is attached to the substrate by the pedal disk (Pd), extends upward along the column and terminates at the oral disk (Od), from which numerous tentacles radiate. B. From the top view, the mouth appears as a slit, which serves as the solitary opening to the gastrovascular cavity (Gv). Note: The siphonoglyphs extend from either end of the mouth (arrowheads). C. Light micrograph of a transverse section of fixed and stained anemone tentacle taken from the approximate location of the doted line in frame B. The tentacle exhibits the holow Gv that extends from the column into each tentacle. D. High magnification of the gastrodermis (Ga) and epidermis (Ep), which are separated by the acellular mesoglea (Mg) (dark blue region indicated by white arrows). Healthy symbionts (Sy) can be seen within symbiosomes in the gastrodermal cells. Scale bars A-B=10mm; C=60 ?m; D=10?m. 26 Figure 2. TEM: Transverse sections of symbiotic Aiptasia palida tentacle tisues. Both tisue layers, the gastrodermis (Ga) and epidermis (Ep), are separated by the acellular mesoglea (Mg). The epidermis and gastrodermis each consist of an irregular epithelium of columnar cells and basal cells. Numerous intact symbionts (Sy) are located within cells of the host gastrodermis. Scale bar=5?m. 27 Gastrodermis Within symbiotic A. palida tisues, columnar gastrodermal cels extend from the mesoglea to the gastrovascular cavity (Fig. 2 and 3), where numerous microvili project from the apical plasma membranes of the cels (Fig. 3, also se Figure 12 and asociated text below). Symbiont cels are located intracelularly within a highly specialized host vacuole caled the ?symbiosome? (Wakefield and Kempf, 2001) (Fig. 3). The host derived membrane of this vacuole and underlying symbiont secretions function as the interface betwen host and symbiont. 28 Figure 3. TEM: Transverse sections of symbiotic gastrodermal tentacle tisues of A. palida. The gastrodermal tisue layer (Ga), extends from the mesoglea to the gastrovascular cavity (Gv). The host cells exhibit a healthy condition with dense cytoplasm and intact nuclei (Hn). Numerous symbiont cells (Sy) displaying healthy appearances and intact nuclei (Sn) were observed within host-derived symbiosomes (black arrowheads). The inset provides higher magnification of the boxed region allowing the symbiosome membrane (double black arrowheads) to be distinguished from the adjacent plasma membrane of the host cell (white arrowhead). Mg, mesoglea; Mv, microvili. Scale bar=2?m. 29 Secretory-gland cels Secretory gland cels were observed in the gastrodermis of A.pallida (Fig. 4). Putative mucus secreting cels are typicaly identified based on the abundant clear or pale inclusions (= vesicles/granules) present in their cytoplasm (Hyman, 1940; Hes et al., 1957; Rose and Burnet, 1968; Westfal et al., 2001). Numerous large pale inclusions were observed in abundance in A. pallida throughout the gastrodermis (Fig. 4A, B). These pale inclusions were observed frequently in regions surrounding symbionts and on occasion appeared to be secretions of symbiont cels (Fig. 4A, B). The overal density and composition of the inclusions being exuded by the symbionts (Fig. 4A, B) was strikingly similar to the numerous other inclusions located within putative gland cels of the gastrodermis (Fig. 4A). Other secretory gland cels were commonly observed throughout the gastrodermis, some of which contained smal iregular electron dense inclusions (Fig. 4A). Mucus-secreting gland cels have been previously described in a variety of cnidarians (Hyman, 1940; Hes et al., 1957; Rose and Burnet, 1968; Westfal et al., 2001), including A. pallida (Westfal et al., 2001) and have a similar appearance to those observed in host gastrodermal cels in this study (Fig. 4A).The pale inclusions (Fig. 4A) observed within putative gland cels of the gastrodermis have the same appearance as the substance that appears to be secreted by some symbionts (Fig. 4A, B). Thus, it is possible that the substance is either translocated from the symbiont cels or synthesized by the host gland cels. However, further investigation is necesary in order to confirm the identity of this substance and its origin. Another type of gland cel that was frequently observed in the gastrodermis contained aggregations of large (~1.5um), round, electron-dense inclusions (Fig. 5). Such granules were first described as ?zymogenic? in a cnidarian gastrodermis by Gauthier (1963) based on their 30 morphological similarity to pancreatic zymogenic granules and their positive enzymatic reactivity. Since then, similar-appearing zymogenic granules have been described in a wide variety of cnidarians (Slautterback and Fawcet; 1959; Haynes and Davis, 69; Vader and Lonning, 1975; Westfal et al., 1997; 2001; Goldberg, 2002; Dandar-Roh et al., 2004) including A. palida (Westfal et al., 1997; 2001). Their general characteristics include that they comprise two-thirds of the volume of a cnidarian gland cel and that they are membrane-bound (Westfal et al., 2001); however, convincing images of a surrounding membrane are not provided in Westfal?s paper (2001) and similar ?zymogen granules? identified by Haynes and Davis (1969) in Hydra viridis are said to lack a surrounding membrane. In order to test the identity of these characteristic cnidarian ?zymogen-like? granules, a recent ultrastructural investigation tested their contents in a symbiotic coral using DMAB-nitrite and Bromophenol blue (Goldberg, 2002). They concluded that the primary composition of the inclusions was similar to that of vertebrate zymogen precursors (Goldberg, 2002). Although no surrounding membrane could be distinguished in asociation with the large dense granules observed in this study, their morphology and organization appeared strikingly similar to previous descriptions in A. pallida (Westfal et al., 1997; 2001). The combined results of the current ultrastructural study with recent ultrastructural-biochemical investigations (Goldberg, 2002) suggest that these large electron-dense inclusions in the gastrodermis of A. pallida are zymogenic granules. However, histochemical testing is necesary to determine the true identity of the contents of these granules in A. pallida. 31 Figure 4. TEM: Transverse sections of symbiotic gastrodermal Aiptasia palida tentacle tisues with gland cells containing various types of vesicular inclusions. A. Numerous putative gland cells containing small electron-dense (arrows) or large pale inclusions (indicated by asterisks) that are commonly present throughout the gastrodermis. B. A symbiont cell appears to be exuding a pale substance (indicated by white arrow and the double asterisks), which has the same appearance as the pale inclusions located in other cells throughout the gastrodermis in 4A (indicated by asterisks).This sugests that the pale inclusions (double asterisks) may be a translocation product released to the host. Nu, nucleus; Sy, symbiont cell. Scale bar A=1?m, B=2?m. 32 Figure 5. TEM: Transverse sections of symbiotic gastrodermal Aiptasia palida tentacle tisues with gland cells containing putative zymogenic granules. Gland cells containing putative zymogenic granules (Zy) were commonly observed in the gastrodermis. Sy, symbiont cell. Scale bar =1?m. Nerve plexus Structures asociated with the nerve net of A. pallida were generaly observed as putative neurites (Fig. 6A-C) that at times clumped together as a bundle of nerve fibers (Fig. 6A,B). Interestingly, the most commonly observed structures that were presumably asociated with the nervous system appeared as densely packed concentric circles within a cel or cel proces 33 located near or adjacent to the mesoglea (Fig. 6D). Due to their abundance within a cel or cel proces, it is not likely that they are axons surrounded by an asociated cel; however, evidence for smal and densely packed neurites has been documented in A. pallida (Westfal, 2002). Alternatively, it is possible that they are dense-core vesicles that have undergone some extraction as a result of procesing for TEM. Cnidarians are the simplest multicelular organisms to posses a nervous system, which is a difuse nerve plexus caled a ?nerve net? (Schick, 1991; Grimelikhuijzen and Westfal, 1995). In most cnidarians the gastrodermal nerve plexus consists of numerous neurites located near the bases of digestive cels (Westfal and Eliot, 2002) that appear similar to those observed in this study (Fig. 6A, B and 9B). The nerve plexus is found just beyond the musculature and transmits signals to various regions throughout the anemone where it contacts other nerve or muscular cels (Schick, 1991; Westfal et al., 2002). In order to cary out this cascade of celular events, several types of cels are necesary, including specialized neurons and ganglion cels (Westfal, 1970; 1973; Anderson and Schwab, 1982). Ganglion cels have been shown interconnect to other ganglion cels through synaptic connections, which suggests that these cels facilitate ?through-conduction of impulses in the nerve net? (Westfal and Eliot, 2002). These cels receive stimuli from sensory cels then proces and transmit the signal to smooth muscle for contraction to occur (Parker, 1919). (Se Epidermis Nerve plexus for more information) 34 Figure 6. TEM: Transverse sections of symbiotic gastrodermal Aiptasia palida tentacle tisues containing putative structures associated with the gastrodermal nerve plexus. A. A region of putative neurites passing amongst other cellular processes. B. Higher magnification of putative neurites (black arrow) showing detail. C. A large cell body containing dense core vesicles (white arrows) and mitochondria (small black arrows) is present at the basal region of the gastrodermis adjacent to the mesoglea (Mg). This cell appeared to narrow at one end (indicated by the double asterisks) in a manner sugestive of a neural projection. D. Putative dense core vesicles were commonly observed in cros- section and appeared to aggregate within a cell or cell process (black arrow) that contains a large lipid droplet (indicated by asterisk) and attaches directly along the mesogleal surface. The inset provides higher magnification of the boxed region showing what appears to be three concentric membranes, the first that of the dense core vesicle and then two additional membranes (white arrowhead and black arrowhead). The presence of these two additional membranes sugests the posibility that this is a process from a neuron that penetrates through an associated cell. Nu, nucleus; Sy, symbiont cell. Scale bars=1?m. 35 Nutritive-muscular cels Host cels that harbored one or more symbionts were the most conspicuous type in the gastrodermis (Fig. 2 and 3). These abundant gastrodermal cels have historicaly been refered to as ?nutritive-muscular? (Hyman, 1940; Brusca and Brusca, 2002) and more commonly, ?epitheliomuscular? (= myoepithelial; Doumenc and Van-Praet, 1987; Schick, 1991; Fautin and Mariscal, 1991; Westfal et al., 2001; Brusca and Brusca, 2002) cels. However, we wil refer to them exclusively as nutritive-muscular cels throughout the remainder of the text 1) in order to diferentiate them from similar but distinct non-symbiont containing cels in the epidermis and 2) to recognize the digestive role of these cels. Nutritive-muscular cels are exclusively found in the gastrodermis and exhibit basal projections that extend circumferentialy adjacent to the mesoglea (Fig. 7). Sparse muscular threads were observed within the basal projections that contribute to the circular smooth muscle of the tentacles (Fig. 7). These structures were further identified as putative myosin (thick) and actin (thin) filaments (Fig. 7) based on their measurements of ~17.5nm and ~7 nm in diameter, respectively, which are both within the expected size ranges of smooth muscle myofilaments in Aiptasia (Amerongen and Peteya, 1976; 1980) (Fig. 7). A similar description of nutritive- muscular cels was made by Hyman (1940) in Hydra as having bases that are ?drawn out into extensions containing a [contractile] myoneme?. ?Myoneme? is a term originaly used to describe contractile fibers in the cytoplasm of protists (Butschli, 1887). Alternatively, Hyman described the muscular component of gastrodermal cels in Anthozoa as cels ?whose bases are drawn out into circular muscle fibers? and fails to describe any basal proceses or myoneme within them; rather describing the base of the cel itself as the muscle fiber. 36 Our observations of the muscular components of nutritive-muscular cels in A. pallida difered from previous descriptions of ?myoneme?-bearing gastrodermals cels in Hydra (Hyman, 1940; Slautterback, 1967; Haynes et al., 1968; Haynes, 1973; Davis, 1973) and those described in other anthozoans (Doumenc and Van-Praet, 1987; Fautin and Mariscal, 1991; Schick, 1991; Westfal et al., 1997; Brusca and Brusca, 2002; Westfal and Eliot, 2002; Tucker et al., 2011). In many of these studies, the organization of the muscular filaments exhibited a rod-like appearance (=myoneme) that has been suggested to result from numerous individual myofilaments stacked together as one contractile unit (Haynes, 1973). In contrast, only sparse single myofilaments were observed as short segments in this study, which may suggest that the filaments are convoluted within the basal proceses in Aiptasia pallida. A previous study suggested that symbiotic anemones contain two distinct types of nutritive-muscular cels in the gastrodermis (Doumenc and Van-Praet, 1987). The first type fits the ?clasical? definition of an?epitheliomuscular? cel by exhibiting highly elongated contractile basal projections and asociated myofilaments (i.e., myoneme) that extend circumferentialy along the mesoglea (Doumenc and Van-Praet, 1987). But the projections and myofilaments of the second type are not as elongated as the previously mentioned cel and these cels often harbor symbiont cels (Doumenc and Van-Praet, 1987). 37 Figure 7. TEM: Transverse sections of symbiotic gastrodermal Aiptasia palida tentacle tisues displaying a nutritive-muscular cell. Numerous nutritive-muscular cells were observed throughout the basal region of the gastrodermis. The processes of nutritive-muscular cell (visible portion outlined by the dashed lines) extend circumferentially (double arrows) and attach directly adjacent to the mesoglea (Mg). The inset provides higher magnification of the boxed region allowing visualization of the myofilaments (white arrowheads) that are present in the cytoplasm. Note: Asterisks indicate putative mucous-containing inclusions. Nu, nucleus; Sy, symbiont cell. Scale bar=2?m; inset:= 1?m. A basal proces clearly asociated with a nutritive-muscular cel-containing symbionts was only observed in one instance (Fig. 8A,B). In this longitudinal section of tentacle, the base of the symbiont containing nutritive-muscular cel could be sen in direct contact with the mesoglea and no micro- or intermediate filaments were evident; unfortunately, the extent of 38 projection of the basal proceses cannot be determined from this single section. Two diferent scenarios are possible. 1) The projections of symbiotic nutritive-muscular cels in A. pallida are reduced in length and musculature compared to the proceses of non-symbiotic nutritive- muscular cels. 2) Gastrodermal cels that are capable of harboring symbionts may lack myofilaments or basal proceses entirely. Without further ultrastructural analysis, the true morphology of these symbiont-containing cels remains uncertain. Mesoglea In anemones, and other cnidarians the mesoglea exists as an extracelular matrix that exhibits ultrastructural and molecular features similar to the extracelular matrix of vertebrates (Tucker et al., 2011). Previous studies revealed that the mesoglea of Hydra contains both striated (Davis and Haynes, 1968) and beaded (Weber and Schmid, 1985) fibrils, which are likely collagen (Zhang et al., 2007) and fibrilin (Megil et al., 2005). The mesogleal layer serves several purposes, including overal structural support and epithelial atachment (Wood, 1961) while facilitating movement of the body wal (Chapman, 1953). Basal proceses of presumably non-symbiotic nutritive-muscular cels were commonly located adjacent to the mesoglea (Fig. 7). Imediate contact betwen nutritive-muscular cels and mesoglea has been reported in other cnidarians (Wood, 1961; Fautin and Mariscal, 1991) and was observed in A. pallida (Fig 8A, B). Longitudinal sectioning of A. pallida tentacles revealed that numerous narow projections of mesoglea also extend deeply betwen adjacent cels and cel proceses in the gastrodermis (Fig. 8C, D). As such, the bases of gastrodermal cels and their proceses appear to be embedded within a web-like system of mesogleal extensions, rather than simply atached to the mesoglea along their basal surface (Fig. 8C, D). 39 Figure 8. TEM: Transverse and longitudinal sections of symbiotic gastrodermal Aiptasia palida tentacle tisues at the region of attachment to the mesoglea. A. Transverse section. Occasionally the attachment of a symbiotic nutritive-muscular cell to the mesoglea (Mg) could be observed. B. Higher magnification of the region where the symbiotic nutritive-muscular cell is directly adjacent to the mesoglea (arrowheads). The lateral processes of this cell appear to be posibly reduced in length compared to the processes of non-symbiont containing nutritive-muscular cells. C. Longitudinal section. Numerous projections of mesoglea extended between and around cells or cell processes as indicated by the white dashed lines. D. At higher magnification substantial portions of mesoglea could be seen surrounding each visible cell process (arrows). One large process containing a large granule (asterisk) was completely surrounded by a prominent layer of mesoglea. The inset shows a higher magnification view of another process surrounded by mesoglea (black arrows). Ep, epidermis; Np, nerve plexus; Nu, nucleus; Sy, symbiont cell. Scale bars=1?m; inset=0.25?m. 40 The mesoglea of A. pallida appeared almost exclusively as an acelular fibrous matrix throughout various regions of the tentacle (Fig. 3-7). However, the mesoglea appeared to be penetrated by adjacent cels on occasion (Fig. 9). Past studies have reported regions of mesoglea traversed by narow cytoplamic proceses (Wood, 1961). In this study, large portions of proceses from adjacent cels protruded into the mesoglea (Fig. 9A,B). Such celular proceses occasionaly appeared to cross the mesoglea and extend betwen cels of the opposite tisue layer (Fig. 9C, D). These extensions represent regions of contact betwen gastrodermis and epidermis. 41 Figure 9. TEM: Transverse sections of symbiotic gastrodermal Aiptasia palida tentacle tisues at the region of attachment to the mesoglea. A. Situations where the mesoglea (Mg) appeared to be interrupted by an adjacent cell were occasionally observed. B. At higher magnification, a cell process penetrates the mesoglea (indicated by the dashed line). The cellular process protruding into the mesoglea, appeared to be a portion of an adjacent gastrodermal cell based on its lack of myofilaments that would be characteristic of epithelia-muscular cells in the epidermis. The penetrating process contains various organelles, including vesicles and mitochondria (black arrows). C. Occasionally, an adjacent gastrodermal cell was observed crosing the mesoglea and extending deeply into the epidermis (double arrows). D. Less commonly, adjacent processes of epithliomuscular cells (indicated by black asterisks) appeared to extend acros and into the mesoglea (indicated by dashed line) and directly contacting the gastrodermis (white arrow). Note: occasionally basal processes exhibited a vacuous appearance, which is likely due to the section cuting through a distal region of the process where the myofilaments no longer extend through the cytoplasm. Putative mucus-inclusions are indicated by white asterisks. Ep, epidermis; Mu, longitudinal smooth muscle; Nu nucleus; Sy, symbiont cell. Scale bars A-C=2 ?m, D=1?m. 42 Epidermis The epidermis is the most complex tisue layer in cnidarians and serves multiple functions for the organism, including defense and prey capture (Fautin and Mariscal, 1991). Several cel types can be found in the epidermis of A. pallida, and the celular organization can be divided into specific regions acording to cel type (Fig. 10). Epitheliomuscular cels The cels located in the closest proximity to the mesoglea (Zone I in Fig. 10) are columnar epitheliomuscular cels (Hyman, 1940; Chapman, 1974, Fautin and Mariscal, 1991). Epitheliomuscular cels are columnar cels that, like the presumptive non-symbiotic nutritive- muscular cels of the gastrodermis, exhibit muscular filaments that appear to be located within prominent longitudinal basal proceses (Fig. 7). However, unlike nutritive-muscular cels, epitheliomuscular cels of the epidermis exhibit numerous myofilaments that are organized into ?bundles' (Fig. 11). These myofilaments make up longitudinal smooth muscle of the epidermis in the tentacles of Aiptasia spp. (Amerongen and Peteya, 1980) that works antagonisticaly against the circular muscle of the gastrodermis (Fautin and Mariscal, 1991). Longitudinal muscle appears stippled in cross section (Fig. 11A) and striated in longitudinal section (Fig. 11B). This appearance results from the presence of thick and thin myofilaments, which likely correspond to the presence of myosin and actin filaments, respectively, both of which have been previously documented in cnidarian muscle (Keough and Sumers, 1976). In the epithelium, the myofilaments of epitheliomuscular cels are more far more dense and abundant than those found in the gastrodermis (Fig. 7), which likely form from numerous myofilaments stacking together as one contractile unit (Haynes, 1973). 43 Densely packed myofilaments concentrated in proceses at the base of the cnidarian epidermis have been generaly described as ?myonemes? in numerous studies (Hyman, 1940; Slautterback, 1967; Davis, 1973; Doumenc and Van-Praet, 1987; Fautin and Mariscal, 1991; Schick, 1991; Westfal et al., 1997; Brusca and Brusca, 2002; Westfal and Eliot, 2002; Tucker et al., 2011). It has also been suggested that the myofilament bundles (=myonemes) may function as independent muscle cels that are separated from adjacent epitheliomuscular cels (Hyman, 1940). In this study, myofilaments were aranged into wel-structured bundles that extended through the longitudinaly oriented proceses located at the bases of epitheliomuscular cels (Fig. 10). Ocasionaly basal proceses of epitheliomuscular cels exhibited a vacuous or ?empty? appearance, which is likely due to the section cutting through a distal region of the cel where the myofilaments no longer extend through the cytoplasm Fig 9A-D). Nerve plexus/ Sensory structures Celular structures asociated with the nerve plexus are typicaly observed directly adjacent and exterior to the bundles of epitheliomuscular cel proceses and their contained myofilaments (Zone I in Fig 10). The nerve plexus of the epidermis is more extensive than the gastrodermis (Westfal and Eliot, 2002; Fig. 6) and appears as a complex network of neurites (Fig. 10 and 11). These neurites can be ganglionic or sensory in origin but cannot be distinguished (Westfal and Eliot, 2002). Clear evidence has been provided in previous investigations of chemical synapses in the epidermal nerve plexus of several anthozoans (Westfal, 1970; Westfal and Eliot, 2002), including A. pallida (Westfal et al., 1998, 1999, 2001; Westfal and Eliot, 2002). 44 However, the overal organization of the nerve net and how muscle contraction is modulated in sea anemone tentacles remains misunderstood (Westfal and Eliot, 2002) and needs further investigation. Figure 10. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues. Several cellular structures were observed throughout the epidermis that can be organized within specific zones based on their general location. In Zone 1, longitudinal smooth muscle (Mu) projects from epitheliomuscular cells directly adjacent to the mesoglea. In Zone 2, the nerve plexus (Np) is located directly adjacent to the muscle cells. Zone 3 consitutes the remaining breadth of the epithelium, where various cnidae (Cn) and other specialized cells are located. Scale bar=3?m. 45 Figure 1. TEM: Transverse and longitudinal sections of symbiotic epidermal Aiptasia palida tentacle tisues displaying longitudinal muscle cells. A. Transverse section showing densely packed myofilaments (My) longitudinally oriented within basal processes of numerous epitheliomuscular cells adjacent to the mesoglea (Mg). High magnifacation of a process allows visualization of individual densely packed thick and thin filaments (inset) The nerve plexus (Np) often appeared as small clear cell processes located immediately adjacent to the muscular processes. B. Longitudinal section showing the orientation of My runing parallel to the neurites of the Np. Scale bars A=1?m; inset=0.25?m, B=1?m. Cnidae Additional cel types were distributed throughout the remaining breadth of the epithelium (Zone II in Fig. 10) where various cnidae and other specialized cels are located (Fig. 10). Cnidae serve as the diagnostic characteristic for the phylum and come in several forms based on function and morphology. Al cnidae are encapsulated in a sheath or tube that is everted upon physical or chemical stimulation (Fautin and Mariscal, 1991). Depending on the type of cnida, the tubule may deliver a toxin, entangle an object, or stick to a prey item (Fautin and Romano, 1997). There are thre major types of cnidae: spirocysts, nematocysts, and ptychocysts, but only the former two have been described in A. pallida (Westfal et al., 1998). Both spirocysts and 46 nematocysts were identified in this study (Fig. 12A,B), but the most prominent type observed in tentacle sections was the spirocyst (Fig. 12A,B). When discharged, these adhere to prey or adjacent substratum and are only known to occur in Anthozoa (Fautin and Mariscal, 1991). Spirocysts appeared as a spiral tubule within an elongated capsule that was housed within a hollow spirocyte (Fig. 12A,B), similar to those described by Westfal (1998). Alternatively, the epidermis of A. pallida also contains nematocysts (Fig. 12A,B), which are the ?stinging? cnidae and typicaly display spines that latch onto prey or predator when everted and inject toxic venom (Fautin and Mariscal, 1991). Nematocysts are easily identified by their thick-waled capsules (Fig. 12B,C), which are basophilic (Kas and Scappatici, 2002) and often impermeable to fixatives and thus, often obstruct the view of the internal spine (Westfal et al., 1998) (Fig. 12B). Cnidae are actualy secreted by the golgi apparatus of specialized cels caled ?cnidoblasts? (Watson, 1988), which were commonly observed in A.palida epithelia (Fig. 12C). Thus, cnidae are not actualy organeles, but, rather, the most complex secretory product known (Fautin and Romano, 1997). Typicaly, a cnidoblast cannot be identified as a nematocyst or spirocyst until a thick capsule becomes visible (Fig. 12C), which is suggestive of further diferentiation into a nematocyst (Westfal et al., 1998). 47 Figure 12. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues displaying various cnidae. A. Numerous nematocysts (white arrows) and spirocysts (black arrows) were observed throughout the epidermis, the latter being the most common. Spirocysts appeared as a spiral tubule within an elongated tube, which was housed within a holow spirocyte (indicated by the double asterisks). B. The thick walled capsules of nematocysts were commonly observed, and occasionally the internal spined tube could also be visualized (small white arrow). C. Cnidoblasts (white arrowheads) were also commonly observed in epidermal tisues. Those developing into nematocysts were often identified based on the presence of the forming thick capsule (inset, white arrow). Numerous cnidoblast nuclei (Nu) were often observed in the region where developing cnidoblasts were present. Zy, zymogen granules. Scale bars =2?m. Sensory structures in the epidermis and gastrodermis Sensory cels and their asociated structures were also occasionaly observed (Zone II in Fig. 10) in both the gastrodermis and epidermis of A. palida (Fig. 13). Sensory cels were most commonly identified based on the presence of an apical cone of stereovili that surrounded a central cilium that extended beyond the microvili of adjacent cels (Fig. 13A). In this study, stereovilar cones and their asociated sensory cilium were most often observed in regions 48 nearby an epidermal nematocyte. This would suggest that they may function in nematocyst discharge in A. pallida as has been shown in other anthozoans (Mariscal and Bigger, 1976; Mariscal, et al. 1978). Apical ciliary cone is the term used to refer to this ciliary-nematocyte receptor complex that is unique to anthozoans and that are conspicuous in their lack of a cnidocil, or hair-like trigger on the nematocyte (Kas and Scappatici, 2002); however, this term is also used to reference non-nematocyte asociated sensory cels (Fautin and Mariscal, 1991). It should be noted that the stereovili of the sensory and sometimes adjacent cels actualy form the cone-like structure that surrounds the central cilium originating from the sensory cel. Within the epidermal nerve plexus of A. palida, various types of neural synapses have been documented, including neuron-spirocyte (Westfal et al., 1999), neuron-nematocyte (Westfal et al., 1998) and neuron-muscular synapses (Westfal and Eliot, 2002). Previous studies have shown that ciliary-nematocyte receptor complexes may be formed entirely by the cilia and microvili of the nematocyte itself or may include projections of supporting cels that surround the nematocyte (Kas and Scappatici, 2002). The later scenario is similar to the sensory cel observed in the epidermis in this study (Fig. 13A), since it appears that at least some of the stereovili that are contributing to the cone-like structure belong to adjacent supporting cels. The cross-sectioned stereovili and central cilium in Fig. 13B displays a similar appearance, where it there is a central large cilium surrounded by several stereovili. These stereovili were unique in their appearance in that they appeared to have remained directly apposed to the central cilium and partialy connected to the adjacent tisue. Similar structures have been previously reported in anthozoans in both sensory cels and phagocytic cels of the gastrodermis (Fautin and Mariscal, 1991). Further sectioning is necesary to determine whether 49 the morphology of the putative ciliary cone in 13B from the gastrodermis is similar to that which we observed in the epidermis. Additionaly, with semi serial sectioning of a complete sensory cel receptor complex it would be possible to determine a significant amount of information about signal transduction in anthozoan sensory cels. Figure 13. TEM: Transverse sections of symbiotic epidermal (A) Aiptasia palida tentacle tisues displaying a sensory cell and associated structures. A. A putative sensory cell (double black arrows) was observed in the epidermis that displayed an apical ciliary cone (large white arrow) that protruded out past the microvili (black arrows) of adjacent cells. This cell extended through the length of the epidermis (double arrows). The apical ends of most epidermal cells displayed numerous microvili (black arrows). B. Several cilia (small white arrows) were also commonly observed at the apical end of many gastrodermal cells along with numerous smaller microvili (black arrows heads). Occasionally, a cros- sectioned modified cilum would be seen surrounded by several directly adjacent stereovili. This appears to be a cros-section of an apical ciliary cone in the gastrodermis. C. In the gastrodermis, many cells displayed numerous microvili (black arrows) as well as what appear to be long thin stereovili (double arrows) that projected into the gastrovascular cavity. Nu, nucleus. Scale bars A=1?m, B=0.5?m 50 Bacterial aggregates Cyst-like aggregations of bacteria were observed in the epidermis of healthy symbiotic A. pallida (Fig. 14) similar to what has previously been described in this species by Palinscar (1989). In both the previous study as wel as the curent investigation, the bacteria appeared packed closely together within a large single vacuole (Fig. 14A). Higher magnification of the bacteria within the cyst revealed that they contained numerous vacuoles and central nucleoids (Fig. 14B). Palinscar and colleagues (1989) determined the identity of identical-looking bacteria to be Vibrio, which have been reported to cause disease outbreaks in sclearactinian anthozoans. Endosymbiotic bacteria have often been documented in cels of a variety of organisms, including cnidarians (Margulis et al., 1978; Thorington et al., 1979). Our observation of bacterial cysts provides further evidence that A. pallida can be ?infected? by bacterial communities; however, whether this asociation is parasitic or mutualistic remains to be determined. Future investigations would benefit by considering the role of bacterial symbioses when measuring the celular or molecular responses of any metazoan. 51 Figure 14. TEM: Transverse sections of symbiotic epidermal Aiptasia palida tentacle tisues displaying a bacterial cyst. A. A bacterial cyst-like aggregate (double arrows) was observed in the epidermis of an otherwise healthy symbiotic anemone. B. Higher magnification of the bacteria within the cyst revealed that they contained numerous vacuoles and central nucleoids (inset). Mg, mesoglea. A. Scale bars A=5?m, B=0.5?m. Conclusions A thorough examination of host symbiotic A. pallida tentacle tisues revealed several interesting results that contradict previous ultrastructural studies. For example, in contrast to earlier descriptions of the mesoglea as an acelular layer to which epithelial cels exhibit simple basal atachment (Gates et al., 1992), our results suggest that the mesoglea functions as a complex network of collagenous substrate that is basaly entangled with the cels and cel proceses of the gastrodermis. The gastrodermal-mesogleal interface has been an important area of interest since Gates et al. (1992) proposed ?host cel detachment? as a potential celular bleaching mechanism. Evidence of host cel detachment was originaly documented in cold-shocked (12 o C) and thermaly stresed (32 o C) Aiptasia pulchela and Pocilopora damnicornis, where it appeared that entire host cels containing intracelular symbionts detached from the mesoglea and were 52 expeled as a result of stres induced-cel adhesion dysfunction. Our observations, suggest that the complex entanglement of gastrodermal cels, cel proceses and the mesoglea observed in symbiotic A. pallida make simple basal separation of symbiont containing cels from the mesoglea an unlikely mechanism for bleaching in this species. The entanglement would make the simple release of cels from the mesoglea very dificult. Alternatively, it is possible that during severe celular stres, symbiotic host cels remain basaly anchored to the mesogleal network, but undergo extensive celular degradation. When structural integrity significantly declines, a portion of the deteriorated host cel may become detached apicaly along with the intracelular symbiont and symbiosome in an apocrine-like apical detachment mechanism and be expeled into the gastrovascular cavity (Chapter 3). During this proces, the remaining host gastrodermal cel maintains its entangled connection to the mesoglea that prevents it from detaching along with the apical portion. The work of Gates et al. (1992) consisted of a few ultrastructural snapshots of degrading tisues of A.pallida and P. damicornis, whereas a subsequent study by Brown et al. (1995) used the light microscope and histological techniques in determining that whole cel detachment occurred. Our results suggest that previous descriptions of host cel detachment (Gates et al., 1992; Brown et al., 1995) may be in need of revision and would benefit from a more in depth ultrastructural re-examination of bleaching in the species and conditions used in those earlier studies. Increased mas mortality bleaching events are predicted to occur in the future (Hoegh- Guldberg, 1999; Donner et al., 2007). Thus, having detailed ultrastructural information of a tractable symbiosis model is critical if we wish to understand the celular mechanisms that underlie the bleaching response. This investigation provides an ultrastructural analysis of tentacle tisues in healthy, unstresed examples of the model symbiotic anemone, A. pallida. The celular 53 information provided here can be used as a baseline against which the health of the Aiptasia model system can be asesed as it is manipulated in the lab or in the field. 54 II. Host autophagic degradation and asociated symbiont loss occur in response to heat stres in the symbiotic anemone, Aiptasia pallida. Abstract Coral bleaching involves the loss of esential, photosynthetic dinoflagelates (Symbiodinium sp.) from host gastrodermal cels in response to temperature and/or light stres conditions. Although numerous potential celular bleaching mechanisms have been proposed, there remains much uncertainty regarding which of them occur during early breakdown of the host-dinoflagelate symbiosis. In this study, transmision electron microscopy was utilized to conduct a detailed examination of symbiotic tisues of the tropical anemone Aiptasia pallida during early stages of host stres. Bleaching was induced by exposing specimens to a stres treatment of 32.5?0.5 o C at 140 ? 5 ?mol photons m -2 s -1 light intensity for 12 h followed by 12 h at 24?0.5 o C in darknes repeated over a 48 h period. Ultrastructural examinations revealed numerous dense, autophagic structures and asociated celular degradation in tentacle tisues after ~12 h of stres treatment. Anemones treated with rapamycin, a known autophagy inducer, exhibited the same ultrastructural characteristics as heat stresed tisues, confirming that the structures observed during heat stres treatment were autophagic. Additionaly, symbionts appeared to be expeled from host cels via an apocrine-like detachment mechanism from the apical ends of autophagic gastrodermal cels. This study provides the first ultrastructural evidence of host autophagic degradation during thermal stres in a cnidarian system and also supports earlier suggestions that autophagy is an active celular mechanism during 55 early stages of bleaching. Introduction Coral refs are one of the most productive and economicaly valuable ecosystems on Earth, providing numerous services to people worldwide (N.O.A.., 2011). However, coral abundance and diversity have dramaticaly declined over the past few decades as a result of growing anthropogenic presures (Donner et al., 2007). Although a wide variety of environmental stresors currently threaten corals, one of the primary causes of large-scale ref degradation is mas mortality bleaching events that have been linked to global climate change and asociated elevated sea surface temperatures (Hughes, 2003; Hoegh-Guldberg et al., 2007). Coral bleaching occurs as a stres response during periods of elevated temperature and iradiation. This results in loss of intracelular dinoflagelates from the genus Symbiodinium (Freudenthal, 1962) from host tisues and/or degradation of symbiont photosynthetic pigments (Hoegh-Guldberg, 1999; Fit et al., 2001), producing a whitened appearance to the host as the underlying calcareous skeleton is exposed through translucent coral tisue. Significant reductions in symbiont densities often lead to reduced growth and fitnes of the host as wel as increased disease susceptibility (Kushmaro et al., 1997; Harvel et al., 1999; Hoegh-Guldberg, 1999). Ultimately, if new symbionts are not re-established within a smal window of time, death of the coral host wil occur (Brown, 1997; Hoegh-Guldberg, 1999). Collapse of the coral-dinoflagelate symbiosis is initiated with increased production of damaging reactive oxygen species (ROS) that occurs during temperature and light stres (Leser, 1996; Franklin et al., 2004). Elevated ROS concentrations have been shown to decrease photosynthetic capability of symbionts through photoinhibition and damage to photosystem I 56 (PSI) (Warner et al., 1999; Leser, 2006). Also, as a result of host mitochondrial membrane damage, high levels of ROS have been shown to difuse into host cel cytoplasm (Ni and Muscatine, 1997; Dunn et al., 2012), where they can directly damage host membranes, proteins (Richier et al., 2005), and DNA (Leser and Farel, 2004). Although coral bleaching has been extensively studied for the past few decades, there remains a great deal of uncertainty regarding which celular events occur during a natural bleaching episode (Weis, 2008). Microscopic examinations of both natural and experimentaly manipulated bleaching tisue have yielded a wide variety of descriptions of potential celular bleaching mechanisms that have been covered in several reviews (Gates et al., 1992; Douglas, 2003; Leser, 2004; Weis, 2008). These include exocytosis (Sten and Muscatine, 1987), host cel detachment (Gates et al., 1992; Sawyer and Muscatine, 2001), and in situ degradation (Taylor, 1973; Brown et al., 1995; Ainsworth and Hoegh-Guldberg, 2008), as wel as multiple forms of ROS-mediated cel death in the form of apoptosis (Dunn et al., 2002; 2004; 2007; Franklin et al., 2004; Strychar et al., 2004; Richier et al., 2006; Ainsworth et al., 2008; Strychar and Samarco, 2009; and Tchernov et al., 2011) and necrosis (Glynn et al., 1985; Dunn et al., 2002; 2004; Strychar et al., 2004; Ainsworth et al., 2008; Strychar and Samarco, 2009). Most recently, an additional form of cel death, resulting from autophagy, has been implicated as an active mechanism of symbiont loss (Dunn et al., 2007; Downs et al., 2009). The autophagic proces functions to isolate old or damaged proteins and/or organeles by isolating them within membrane-bound autophagosomes and degrading the contents for recycling. An alternative form of autophagy, caled ?symbiophagy?, has been reported in a thermaly-stresed coral, Pocilopora damicornis, whereby the symbionts themselves were digested within their host-derived symbiosomes (Downs et al., 2009). 57 The variation among previously described celular bleaching investigations likely results from several factors. First, microscopical observations have been made in numerous diferent anthozoan host-symbiont complements under a wide range of environmental bleaching conditions, including methods such as cold shock (e.g., Gates et al., 1992), which is les commonly observed in the natural environment. Second, much emphasis has been placed on the stres response of the symbiont during bleaching rather than that of the host (Baird et al., 2008; Weis, 2008). However, increasing evidence suggests that the host celular response plays an important role in the bleaching proces (Baird et al., 2008) and may precede that of the symbiont (Ainsworth et al., 2008; Dunn et al., 2012). Third, a majority of previous investigations have relied on ?snapshots? in time of stresed tisues (often without comparison to controls) as a basis for their histological interpretations, rather than a sequential examination of bleaching tisues throughout the proces. Thus, much of our current information has been gained from brief glimpses of the bleaching response, particularly focusing on late stages in the proces. As a result of these limitations, our current understanding of the celular mechanisms of bleaching is far from complete (Weis, 2008). It has been suggested that in order to acelerate our understanding of celular bleaching, a tractable model species, such as the symbiotic anemone, Aiptasia pallida (Agasiz in Veril, 1864), should be utilized in experimental studies under naturaly relevant environmental conditions (Weis et al., 2008). In this study, we aim to elucidate some of the mising pieces of information surrounding the celular bleaching proces by conducting a thorough ultrastructural examination of host anthozoan tisues at multiple stages of the early stres response using the symbiotic anemone, Aiptasia pallida. Heat stres conditions were used to induce gradual bleaching, and ultrastructural changes were systematicaly analyzed and compared to unstresed, healthy control 58 animals. Results from this study provide a beter understanding of how the anthozoan host responds during the early stages of a bleaching event and examines the role of autophagy in the host stres response during symbiont loss. Materials and Methods Culture conditions Aiptasia palida harboring symbionts typed as Clade A4 Symbiodinium (Santos et al., 2002; Scott Santos, pers. comm.) were collected in the Florida Keys and maintained in artificial seawater (Ref Crystals) at 28-30 ppt salinity. Anemones were kept in two 150 gal tanks at 24?1?C. The bottom of each tank, where most anemones were located, was ~ 1 m below a light source that covered the length of each tank. The light source consisted of two fluorescent light fixtures per tank that were each equipped with two 32W bulbs (Philips F32T8/TL841) producing a 50?5 ?mol photons m -2 s -1 iradiance at the bottoms of the tanks. Lights were set on a 12:12 h light/dark regime. The anemones were fed freshly hatched Artemia naupli thre times per wek. Field seawater temperature determination In order to approximate ecologicaly relevant bleaching conditions in this study, diurnal changes in surface water temperature were measured at the collection site in Key Largo, Florida using a standard ethanol thermometer at six regular daily intervals for 5 days. Average temperature values were calculated at 30?1?C during the day and 24?1?C at night. Stresed A. pallida were subjected to similar daily temperature changes with the daytime temperature adjusted to a value that would cause bleaching (as described below). 59 Bleaching conditions Twelve medium sized anemones were placed in identical, individual dishes that contained ~200 ml 0.45 ?m Milipore-filtered artificial aquarium sea water (MFAW) and alowed to aclimate for ~5 days at the ambient room temperature of 24?1 o C with 50?5 ?mol photons m -2 s -1 ambient light intensity. Water in these dishes was changed daily and the anemones were fed once during the first 3 days. They were then held unfed for 48 h prior to the beginning of the experiments. Six anemones were then randomly selected and transfered in their containers to an incubator at 32.5?0.5 o C and placed ~14 cm beneath two light fixtures each equipped with two 20W fluorescent bulbs (GE Ecolux F20T12/G50-ECO) that emited 140?5 ?mol photons m -2 s -1 light intensity at the level of the anemones. The anemones were held in the incubator for 12 h and then removed to a dark, wel-ventilated box on the lab bench for 12 h in darknes at 24?1 o C. The incubator and dark box treatments were then repeated over the following 24 h for a total treatment time of 48 h. It took approximately 2.5 h for the 24?1 o C dishes to warm to 32.5?0.5 o C in the incubator under these conditions. This study primarily focuses on the efects of heat stres. There was a smal diference in light intensity betwen the anemone culture tanks (50?5 ?mol photons m -2 s -1) and the incubator (140?5 ?mol photons m -2 s - 1 ); however, this is wel within the field light intensities (0 to > 1000 ?mol photons m -2 s -1 ) that these symbiotic anemones experience and was not considered to be a stresor in our experiments. In order to test for efects of any ambient environmental factors, six additional anemones aclimated as described above were randomly selected as controls (no stres treatment) and were placed in the same type of dishes used for stresed anemones at 24?1?C with 50?5 ?mol photons m -2 s -1 iradiance for 12 h daily folowed by 12 h in darknes over a 48 h period in a subsequent 60 experiment. In al cases water was changed daily and anemones were not fed during the 48 h treatment period. Biochemical induction of autophagy Anemones were maintained in their standard culture conditions at 24?1 o C and 50?5 ?mol photons m -2 s -1 iradiance with a 12:12 h light/dark regime. Several symbiotic anemones (n=6) were exposed to one treatment of 25 ?M rapamycin (Sigma-Aldrich) in a solution of 1% DMSO in MFAW for 12 h (Dunn et al. 2007). Control anemones (n=6) were exposed to 1% DMSO in MFAW for 12 h under identical conditions. Fixation, embedment and sectioning for TEM Treatment anemones exposed to heat stres (0, 12, 24, or 48 h) or to 24 ?M rapamycin (12 h) and control anemones exposed to no stres (0 and 48 h) and to 12 h of 1% DMSO were al prepared for TEM following the same protocol. First they were relaxed by removing the seawater from each anemone container and replacing it with ~300 mL high Mg/low Ca seawater (Audesirk and Audesirk 1980) for 30 mins, followed by the addition of ~ 2 mL chlorotone saturated seawater for 30 mins. Then a subset of tentacles (n=6) was clipped from each of 3 randomly selected anemones at each time point (stres treatments and controls). Tisues were fixed using methods similar to the protocol of Caroll and Kempf (1994) in Milonig?s phosphate buffered, 2.5% gluteraldehyde fixative solution for 1 h followed by rinses with a 1:1 0.34M NaCl/Milonig?s phosphate buffer solution. The tentacles were secondarily fixed in a 1.25% NaHCO 3 buffered, 2% OsO 4 solution for 1 h. Following secondary fixation, tentacles were rinsed thre times in 1.25% NaHCO 3 buffer , dehydrated through an ethanol series to 100% 61 ethanol, transfered through 3 changes of propylene oxide, and infiltrated and embedded using EMbed 812 resin (Electron Microscopy Sciences). Al treatments were caried out at room temperature (24?1?C). Embedded tentacles were sectioned (~60nm) using a diamond knife (Diatome) on a Reichert-Jung Ultracut E microtome. Al sections were stained with uranyl acetate and lead citrate for transmision electron microscopy (TEM). Tisues were visualized and photographed in sections from mid-tentacle length and around the circumference of each tentacle using a Zeis EM 10C 10CR transmision electron microscope. Negatives from TEM were scanned as positives on an Epson Perfection 3200 Photo flatbed scanner at 1200 dpi and contrast and levels were adjusted using Adobe Photoshop 8.0 (n=142). A detailed ultrastructural examination of healthy, symbiotic, control A. pallida tentacle tisues from 0 and 48 h provided baseline data that were used for comparison to tisues exposed to various time periods of heat stres or to rapamycin treatment. Such comparisons alowed ultrastructural variations in stres and rapamycin treated tisues to be identified. The possible occurrence of cel death mechanisms, such as apoptosis and necrosis, was determined based on celular ultrastructural characteristics specific for these mechanisms as reported for A. pallida and its intracelular symbionts (Dunn et al., 2002; 2004). Cels were considered apoptotic when they exhibited condensed chromatin with cel shrinkage and/or membrane blebbing, and in contrast, necrotic when the presence of lysed celular material and/or cel sweling was observed (Dunn et al., 2002; 2004). Additionaly, cels were determined to be autophagic if autophagy- derived structures (i.e. autophagosomes, autophagolysosomes, tertiary lysosomes, residual bodies) similar to those previously described in vertebrate (Hand, 1970; Fawcet, 1981; Holtzman, 1989; Jing and Tang, 1999; Nixon, 2007) and invertebrate (Kov et al., 2000; Rost- Roszkowska et al., 2008) tisues were observed in either host or symbiont cels. These structures 62 are commonly identified by the presence of celular material at various stages of degradation within their membranes (Holtzman, 1989; Jing and Tang, 1999; Levine and Yuan, 2005; Nixon, 2007; Rost-Roszkowska et al., 2008). Algal cel counts In order to quantify symbiont loss resulting from stres-induced bleaching, expeled Symbiodinium cels were collected from both control and stresed anemone containers (n=3) after 48 h of treatment. For each anemone the collected algae were centrifuged, and then re- suspended in 250 ml of 0.45 ?m MFAW. Cels were then counted with a Neubauer hemacytometer (acording to Perez and Weis, 2006) and the average number of algal cels that were expeled per anemone after 48 h was calculated for controls and heat stres treatment anemones. Statistical analysis Significance of the diference in average number of expeled Symbiodinium cels/anemone after 48 h for both control and heat stres anemones was determined using a t-test at significance level of 5% (alpha=0.05) using SAS 9.2 software. 63 Results and Discusion Examination of symbiotic anemone tentacle tisues prior to the heat stres treatment (at 0 h) revealed a healthy overal appearance (Fig. 15A-C) identical to that of control anemones at 0 and 48 h (Fig. 15D-E). In general, unstresed host tisues consisted of intact gastrodermal and epidermal cel layers separated by a thin layer of acelular mesoglea (Fig. 15A). The epithelial cels that make up each layer consistently exhibited a characteristicaly healthy celular composition (Fautin and Mariscal, 1991; Shick, 1991) with a normal distribution of organele types (e.g. nucleus, mitochondria, etc.) surrounded by abundant dense cytoplasm. In the cytoplasm of the epidermis, nematocyst capsules were often observed, appearing as capsule membrane surrounding dark, oval-shaped structures of varying size depending on the plane of section (Fig. 15B). In the gastrodermis, numerous epithelial cels harbored intact, healthy Symbiodinium cels within a host derived symbiosome membrane (Fig. 15A, C, E) (Wakefield and Kempf, 2001). In both tisue layers, electron dense lipid droplets were occasionaly observed, which were easily recognized by their characteristic smooth and approximate circular or oval shape, homogeneous osmophilic appearance, and lack of a surrounding membrane (Fig. 15B). There was litle or no evidence of apoptotic, necrotic or autophagic activity present in host or symbiont cels from control anemones or from treatment anemones prior to stres. However, in situ degradation of symbionts was occasionaly observed in some sections. 64 Figure 15: TEM: Transverse sections of control and unstressed treatment (t=0) symbiotic Aiptasia pallida tisues. A-C) Healthy unstressed tisues (0 h of heat treatment): A) Host cells exhibit a normal appearance with abundant dense cytoplasm in both gastrodermis and epidermis. In the cytoplasm, electron dense circular or oval structures were occasionally observed (boxed regions). Numerous intact Symbiodinium cells (Sy) were observed within host gastrodermal cells. B) Higher magnification of the epidermal tisue layer, showing homogeneous osmophilic appearance and regular shape of lipid droplets (left panel) and nematocyst capsules (right panel). C) Higher magnification of the gastrodermal tisue layer (large, right boxed region in A) showing the healthy condition of host cells. Two dark structures are indicated (white arrowheads with black border) that are likely small lipid droplets. D and E) Control (48 h at ambient temperature and irradiation) tisues for comparison: D - Epidermis and E - Gastrodermis. nu = host cell nucleus. Scale bars = 5 ?m (A) and 2 ?m (B-E). 65 The ultrastructure of anemone tisues changed dramaticaly after exposure to the heat stres treatment described above. Examination of stresed tisues starting at 12 h of treatment revealehd that both the gastrodermis and epidermis contained increasing numbers of electron dense, iregularly shaped cytoplasmic bodies that exhibited a heterogeneous appearance (Fig. 16) and were easily distinguishable from smooth, homogeneous lipid droplets. At higher magnification, it became apparent that these dense structures contained varying amounts of acumulated and/or partialy digested cytoplasmic material (Fig. 16B-C), which is a characteristic of active autophagic structures (APSs). Other cel death activities, including apoptosis and necrosis (se methods) were infrequently observed in host and/or symbiont cels throughout the duration of heat stres. Additionaly, no evidence of exocytosis of symbionts from host gastrodermal cels or of host cel detachment was observed; however, as in the controls (se above) in situ degradation of symbionts was occasionaly observed in treatment tisues (se below). 66 Figure 16: TEM: Transverse sections of symbiotic Aiptasia palida tisues after 48 h of exposure to heat stress. A) Host tisues exhibit numerous dense APSs throughout both the gastrodermis and epidermis. B) Higher magnification of the epidermis where sequestered and partially digested cellular materials can be seen within large APSs (indicated with asterisks). C) Higher magnification of the gastrodermal tisue layer with smaller but similar APSs (indicated with asterisks) that also have a heterogeneous appearance. Nucleoli are indicated in B and C (black arrow with white outline) within host nuclei for comparison. Scale bars = 5 ?m (A) and 2 ?m (B, C). The observed APSs varied in their appearance as they progresed through several stages of development, (Fig. 17). These include 1) initial formation of a characteristic ?cup-like? pre- autophagosome structure (Fig. 17A), 2) sealing of pre-autophagosomes and early sequestration of surrounding cytoplasmic and organelar materials for digestion (Fig. 17B), and 3) further sequestration/digestion of contents and retention of indigestible material forming electron dense APSs (Fig. 17B-D). Autophagic degradation within cels and their proceses resulted in regions with sparse cytoplasm and numerous dense APSs (Fig. 17D). Both ?young? and ?mature? 67 (electron dense) APSs were often observed in what appeared to be the active sequestration of celular materials (note invagination of several APSs - double asterisks, in Fig. 17B-D). Figure 17: TEM: Transverse sections of symbiotic Aiptasia palida tisues after 48 h heat stress showing various stages of APS formation (indicated with 1 or more white asterisks). A) Induction: Pre- autophagosome formation via ?cup-like? structure (double arrows point to lip of cup) located nearby a mature APS (asterisk). B) Sequestration: Surrounding cellular materials are sequestered into an autophagolysosome (double asterisks) where they are actively degraded. Other nearby autophagosomes (single asterisks) are also indicated. C) Retention and further sequestration: APSs can vary in size and density depending on the type and amount of materials that are retained and the extent of digestive breakdown of sequestered materials. Note: Early APSs often appear less dense (black asterisks with white outline) D) Formation of large, dense APSs that result from accumulation and digestion of cellular material. Cytoplasmic degradation is often observed as regions of ?cleared? cytoplasm where numerous APSs and suspended organelles (black arrowheads) are found (e.g., regions 1, 2, 3, 4). Double asterisks indicate APSs that appear to be actively sequestering cytoplasmic or organellar material. Scale bars = 0.5?m (A) and 1?m (B-C). 68 Biochemical induction of autophagy In order to confirm the role of autophagy in APS formation and subsequent celular degradation during heat stres, several symbiotic anemones were exposed to 25 ?M rapamycin in seawater containing 1% DMSO for 12 h (Dunn et al. 2007) (Fig. 18) and the ultrastructural appearance of their tisues was compared to that of anemones experiencing heat stres (Fig 16). Ultrastructural examination of tentacle tisues from rapamycin treated anemones (Fig. 18E-F) revealed a strikingly similar celular response to that sen in heat stres-induced anemones (Fig. 18B-C) with numerous APSs and highly degraded celular regions present when compared to controls (Fig. 18A,D). As was the case with heat stresed anemones, a significant amount of expeled algae was observed at the bottom of each rapamycin treatment anemone container. Al treatment anemones exhibited reduced brown coloration (presumably resulting from symbiont loss) after 12 h of rapamycin incubation when compared to controls. 69 Figure 18: TEM: Transverse sections of tentacles at midlength showing autophagic structure formation in the gastrodermis of symbiotic anemones. A) Control tisues after 0 h of heat stress display healthy ultrastructural appearance with few to no visible autophagic structures, B) After 48 h of heat stress tisues reveal numerous dense autophagic structures, C) Higher magnification of the boxed region shows detail of the APSs (asterisks). D) Control tisues exposed to 1% DMSO for12 h (no rapamycin) exhibit few to no APSs with no degradation, E) Tisues exposed to 12 h of 25 ?M rapamycin result in the presence of numerous dark APSs, F) Higher magnification of the boxed region shows detail of the APSs (asterisks). Sy = symbiont; Mg = mesoglea. Scale bars = 5?m (A, B, D, E) and 2?m (C, F). Celular bleaching mechanisms After ? 12 h of heat stres, tisues often underwent excesive autophagic degradation resulting in loss of significant amounts of cytoplasmic material from either regions of cels and their proceses or from entire cels (Fig. 18A-C). This degradation likely resulted from sequestration and subsequent digestion of celular material by APSs, which were often observed within or near degraded celular regions (Fig. 17D, 19A-D). Gastrodermal cels that harbored Symbiodinium cels frequently exhibited deterioration of their cytoplasm at the apical region of 70 the host cel betwen the symbiont and gastrovascular cavity (GVC) (Fig. 19A) and occasionaly throughout the majority of the cel (Fig. 19B-C). Symbionts generaly remained healthy in appearance (based on work of Taylor, 1968) with intact chloroplasts and other organeles throughout the duration of both the heat stres (Fig. 16A and 18B) and rapamycin (Fig. 18E) treatments. However, symbionts found within degraded host cels often appeared to have migrated or been moved toward the apical region of the host cel (Fig. 19A-B). Apicaly positioned symbionts were commonly observed bulging out into the GVC along with surrounding host cel cytoplasm and plasma membrane (Fig. 19B). These symbionts and surrounding host cel cytoplasm and plasma membrane appeared to be actively detaching from apical portions of host cels in what we describe as an apocrine-like manner, since both "secretion" (the symbiont) as wel as a portion of the host cel are lost from the gastrodermal cel (Fig. 19D-F). During this detachment proces (hereafter refered to as ?apical detachment?), the separating, symbiont-containing bleb initialy maintained a connection to th e host cel via thin strands of host cytoplasm and plasma membrane (Fig. 19D) before apparent release into the GVC (Fig. 19E). Although fully detached symbionts remained surrounded by a thin layer of host cytoplasmic and membranous material (Fig. 19E), this host material appeared to be shed from symbionts as they moved further into the GVC (Fig. 19F). Additionaly, degraded symbionts were occasionaly observed in heat stresed tisues (Fig. 19E). 71 Figure 19: TEM: Transverse sections of symbiotic Aiptasia palida after ? 12 h heat stress showing apparent apical detachment of symbiont containing blebs from autophagically degraded host gastrodermal cells (double white arrows with black border). Several large APSs are indicated with asterisks. A) After 48 h of treatment autophagic degradation results in sparse cytoplasm between symbiont and apical edge of host cell (double white arrows). B) After 12 h of treatment an apparently healthy symbiont has relocated toward the apical end of an extensively degraded host cell, where it bulges out into the gastrovascular cavity within the host plasma membrane. C) Higher magnification of boxed area in B, where autophagically degraded cytoplasm is apparent in basal region of a host cell. Note numerous large APSs are located within the heavily deteriorated region where only very sparse cytoplasm remains. D) After 24 h of heat stress treatment a symbiont and surrounding host cytoplasm and plasma membrane with microvili is seen bulging into the GVC, as it appears to be completing its separation from the apical end of an autophagically degraded host cell in an apocrine-like manner. E) After 48 h of treatment an apparent fuly detached bleb of symbiont containing host cell cytoplasm and associated plasma membrane floats free within the gastrovascular cavity. Inset shows higher magnification of host membrane (black arrows), underlying symbiosome structures, and symbiont cell wall (black arrowhead). F) After 48 h of treatment several symbionts are seen at various stages of apical detachment. The furthest symbiont from its detachment site has only small remnants of surrounding host cytoplasm and membranous components. Inset shows higher magnification of remaining host material (black arrows) and symbiont cell wall (black arrowhead). Scale bars = 5 ?m (A, B, D-F) and 2?m (C). De = degrading symbiont; dSy = dividing symbiont; Mg = mesoglea ; Sy = symbiont. 72 After ~12 h of heat stres, anemones exhibited a noticeable loss of brown coloration, indicating that bleaching was actively occurring. Average symbiont loss was quantified in both control and heat-treated anemones after 48 h, the later of which exhibited a significant 10-fold increase in the number of expeled cels per anemone (Fig. 20). Figure 20: Average number of expelled Symbiodinium cells per anemone after 48 h of either heat stress or control treatment. (t-test, p < 0.05, n=3). 73 Conclusions In this study, an abundance of autophagic structures (APSs) was observed within A. pallida tentacle tisues after exposure to heat stres. These were similar in structural appearance to those observed after 12 h of exposure to the autophagy inducer, rapamycin. Since rapamycin has previously been employed to biochemicaly induce autophagy in a variety of taxa, including Hydra (Chera et al., 2009) and A. pallida (Dunn et al., 2007), this provides ultrastructural evidence that elevated autophagic activity occurs as a celular stres response in A. pallida during early stages of bleaching. The autophagic proces involves sequestration and intracelular breakdown of long-lived proteins and damaged organeles for recycling (Levine and Yuan, 2005; Mizushima and Klionsky, 2007). This occurs through a series of developmental stages, which were observed in heat stresed anemone tisues (Fig. 21). These begin with construction of a membranous pre- autophagosomal structure that seals around cytoplasmic material to form a complete ?autophagosome? (Holtzman, 1989; Jing and Tang, 1999). At some point, autophagosomes must fuse with a primary lysosome in order to gain the necesary enzymes to degrade the sequestered contents (Fawcet, 1981; Jing and Tang, 1999) and are then generaly termed ?autophagolysosomes? (Holtzman, 1989). The APSs continue to sequester cytoplasmic materials as enzymes begin to degrade the contents, eventualy leaving only electron dense, heterogeneous, indigestible materials within the autophagosome membrane (Fawcet, 1981; Holtzman, 1989). Interestingly, in tisues of A. palida, even excedingly electron dense APSs appeared to continue to sequester additional celular material (Fig. 21). Variation in the density of APSs was observed, which likely correlated with the level of maturation (i.e. material degradation and retention) that each structure was experiencing at the time of fixation. 74 Figure 21: Stages of autophagy begining with formation of the cup-like pre-autophagosome that extends around portions of cellular material (designated as gray circles) and eventually seals forming a double membrane bound autophagosome. Upon fusion with a lysosome the structures are often referred to as an autophagolysosome. Here, the iner membrane is quickly disolved as lysosomal enzymes begin to degrade the sequestered cellular contents. Autophagolysosomes continue to sequester and degrade surrounding cellular material (early degradation stage) until only dense indigestible materials remain (late degradation stage- often referred to as a residual body). Autophagosomes and autophagolysosomes are colectively referred to as autophagic structures (APSs). Since significant numbers of APSs were found in both symbiotic gastrodermal and non- symbiotic epidermal tisues, it would appear that stres induced-autophagy is primarily a host- mediated celular activity. However, further investigation is necesary to determine any contributive role that the symbiont might have during this autophagic stres response (se comments on ROS below). Autophagy-like celular activities have been previously implicated in the bleaching proces. One study conducted by Dunn et al. (2007) provided biochemical evidence of both autophagic and apoptotic activity during heat stres-induced bleaching in A. palida. The authors suggested that no single cel death pathway could be implicated during the bleaching proces and that both mechanisms contributed to the loss of symbionts. Their results suggested that apoptosis and autophagy were interconnected, such that when one is inhibited, the other is induced. However, the manner in which these two celular proceses progresed temporaly throughout the bleaching episode was not described, and no ultrastructural evidence of the occurrence of 75 autophagy was provided. Additionaly, no evidence of symbiont digestion or ?symbiophagy? (per Downs et al., 2009) was observed in our study. In this investigation, nearly al symbionts displayed a healthy appearance throughout the heat stres treatment, as evidenced by intact thylakoids in the chloroplasts as wel as overal ?normal? ultrastructure (per Taylor, 1968). This may be due to the fact that the type A4 Symbiodinium strains present in Aiptasia from the Florida Keys have been previously shown to exhibit higher tolerance to temperature stres (Goulet et al., 2005) and thus, may beter maintain its celular structure under heat stres conditions. Additionaly, in situ degradation of symbionts was occasionaly observed in both heat stresed and control tisues in this study, suggesting that symbiont degradation may function as a naturaly occurring means of population control rather than as a stres response. Autophagic activity is an esential mechanism for cel survival and homeostasis (Ohsumi and Mizushima, 2003), but typicaly remains suppresed to low levels in most cels (Levine and Yuan, 2005). However, under various stres conditions, such as starvation (Scott et al., 2004) or heat stres (Prasad et al., 2007; Oberley et al., 2008; Swanlund et al., 2008), autophagy is often upregulated as a pro-survival response by maintaining mitochondrial ATP energy production through the recycling of nutrients (Levine and Yuan, 2005). Alternatively, autophagy has also been implicated as a cel death mechanism (Xue et al., 2001), whereby excesively damaged cels are removed from afected tisues (Kourtis and Tavernarakis, 2009). Autophagy and other forms of cel death can be triggered by production of ROS (Scherz- Shouval et al., 2007; Chen and Gibson, 2008), which has been reported to induce cnidarian bleaching (Franklin et al., 2004; Perez and Weis, 2006). If the amount of celular stres is minimal, then intracelular ROS levels remain low, and autophagy would be maintained at normal levels, thus, promoting survival. Alternatively, exposure to high temperature would 76 eventualy result in excesive ROS levels in the host (Ni and Muscatine, 1997; Richier et al., 2006) that could result in damage to cytoplasmic organeles. The damaged intracelular debris would likely induce autophagic pathways and numerous APSs would be formed. These APSs would extensively digest surrounding cytoplasmic material and organeles, similar to the results we observed after 12 - 48 h of heat stres in A. pallida. Heavily stresed cels that become damaged beyond repair are likely to undergo autophagic cel death as they atempt to restore and maintain tisue homeostasis. This may facilitate bleaching during early stages of the stres response. Thus, ROS production by symbionts may act to facilitate host-mediated autophagy. Expulsion of symbionts from gastrodermal cels via apical detachment appeared to be asociated with autophagic degradation of cels. This may be a bleaching response that occurs when autophagy eliminates large amounts of cytoplasm and organeles from gastrodermal cels and both structural integrity and stability are systematicaly lost. As a result, intracelular symbionts may either migrate or be moved toward the apical end of the host cel, where they are eventualy expeled along with surrounding host celular material via an apocrine-like mechanism. A similar mechanism for symbiont loss, caled ?budding?, was suggested by Glider (1983) as a method of symbiont population control in non-stresed A. pallida. Similarly, Gates et al. (1992) suggested that "pinching of" might be a mechanism for symbiont release during bleaching, though they did not observe this proces nor was it asociated with autophagy or any similar cel death-related activity. Since symbionts exhibiting litle or no asociated host cytoplasm or plasma membrane were commonly observed in the GVC, but no evidence of exocytosis of symbionts was observed, it is possible that digestive enzymes or enzymes released from dying cels may quickly degrade the host-derived material that surrounds symbionts shed via apical detachment. 77 Although the majority of previous celular bleaching studies have documented high levels of apoptotic or necrotic activity in both host and symbiont during heat stres (Dunn et al., 2002, 2004, 2007; Franklin et al., 2004; Strychar et al., 2004; Richier et al., 2006; Ainsworth et al., 2008; Strychar and Samarco, 2009; Tchernov et al., 2011), the most prevalent celular activity observed throughout the duration of the present study was host cel autophagy. These ultrastructural findings provide further evidence to support the suggestion of Dunn et al. (2007), that apoptosis is mostly suppresed while autophagic activity remains elevated during heat stres. Further investigation is necesary to determine whether autophagic cel death is replaced by apoptosis later in the stres response. The results of this current investigation support the hypothesis that in A. pallida and perhaps other anthozoan species, elevated autophagic activity and autophagic cel death function as an early response to heat stres. Although these results suggest that autophagy may occur as the first celular stres response during moderate to acute stres conditions (32.5 o C), it is possible that under more dramatic conditions, such as exposure to 1) excedingly high temperatures or 2) longer periods of stres, that diferent cel death pathways may be initiated as has been previously suggested (Dunn et al. 2002, 2004). Mas mortality bleaching events are predicted to increase in frequency and intensity over the next few decades (Hoegh-Guldberg, 1999; Donner et al., 2007). Therefore, it is critical to advance our current knowledge of the bleaching proces by providing a complete analysis of how breakdown in the symbiotic asociation occurs. The results of this study provide a beter understanding of the celular stres responses in the common Symbiodinium symbiosis model, A. pallida, through a simulation of more natural bleaching conditions and a detailed examination of host tisues at multiple time points in heat stresed and control anemones. Results from this study 78 present the first ultrastructural evidence of autophagic digestion of host tisues during thermal stres in a cnidarian system. Additionaly, we suggest that elevated autophagic activity may induce the loss of symbionts via apical detachment from degraded host cels. Further studies are needed both to verify this bleaching mechanism and to determine other celular changes that may occur during later periods of heat stres-induced bleaching. 79 IV. Autophagic activity occurs as a host-derived mechanism in the symbiotic anemone, Aiptasia pallida, during heat stres-induced bleaching Abstract The proces known as coral bleaching is characterized by the loss of esential, photosynthetic dinoflagelates, Symbiodinium spp., from host tisues in response to elevated temperature and/or light stres. Previous studies have implicated the involvement of several possible methods of cel death that underlie the cnidarian bleaching proces, most recently highlighting the possible role of autophagic activity. In this study, transmision electron microscopy was utilized to conduct a detailed examination of symbiotic tisues of the anemone, Aiptasia pallida, during early stages of stres-induced bleaching. Both symbiotic and aposymbiotic anemones were exposed to a stres treatment of 32.5?0.5 o C at 140 ? 5 ?mol photons m -2 s -1 PAR light intensity for 12 h followed by 12 h at 24?0.5 o C in darknes during two consecutive 24 h cycles for a total of 48 h. In stresed symbiotic anemones, significantly higher numbers of autophagic structures (APSs) and asociated celular degradation were observed in both host gastrodermis and epidermis. Stresed aposymbiotic anemones exhibited significantly elevated autophagic activity in the gastrodermis, suggesting that autophagy occurs primarily as the result of a host-derived celular pathway. Treatment of anemones with the known autophagy inducer, rapamycin, resulted in significant increases in APSs in both epidermis and gastrodermis with similar ultrastructural appearance to that of symbiotic heat stresed tisues. These results confirm previous suggestions that the structures observed during the heat stres treatment are autophagic. This study provides the first quantitative evidence in a symbiotic cnidarian that 80 autophagic activity is elevated during the initial 48 h of heat stres that results in bleaching. Introduction The coral bleaching proces is characterized by the loss of esential, photosynthetic dinoflagelates of the genus Symbiodinium (Freudenthal, 1962) from gastrodermal tisues of corals or other symbiotic cnidarians. This phenomenon can occur in response to a variety of environmental stresors; however, elevated heat and/or light conditions are most commonly asociated with episodes of mas mortality bleaching. During elevated temperature and light conditions, the cnidarian-dinoflagelate symbiosis breaks down through a complex series of celular interactions which are initiated when the stres threshold of either or both member(s) is surpased and excesive amounts of damaging reactive oxygen species (ROS) are produced by the symbiont (Franklin et al., 2004; Leser, 1996) and/or host cels (Dunn et al., 2012; Dykens et al., 1992; Ni and Muscatine, 1997). If the rate of ROS generation exceds the rate of detoxification, then acumulation of ROS may result in oxidative damage to celular structure, loss of cel function, and ultimately death of the afected cel (Chen and Gibson, 2008; Leser, 1997; Perez and Weis, 2006; Sherz-Shouval et al., 2007). Recent investigations suggest that such ROS-mediated stres is the underlying cause of symbiont loss during bleaching (Franklin et al., 2004; Leser, 2007; Perez and Weis, 2006). Several celular bleaching mechanisms have been proposed that implicate the involvement of cel death-related activities in the host and/or symbiont. Such mechanisms include apoptosis (Ainsworth et al., 2008; Dunn et al., 2002; 2004; 2007; Franklin et al., 2004; Richier, et al., 2006; Strychar et al., 2004; Strychar and Samarco, 2009; Tchernov, et al., 2011), necrosis (Ainsworth et al., 2008; Dunn et al., 2002; 2004; Glynn, 1985; Strychar et al., 81 2004; Strychar and Samarco, 2009), and autophagy or autophagy-like proceses (Dunn et al., 2007; Downs, et al., 2009; Chapter 3). Although autophagic activity has been wel documented in both vertebrate (Fawcet, 1981; Hand, 1970; Holtzman, 1989; Jing and Tang, 1999; Nixon, 2007) and invertebrate (Kov et al., 2000; Rost-Roszkowska et al., 2008) tisues, it has only recently been identified in stresed symbiotic cnidarians (Dunn et al., 2007; Chapter 3). This autophagic proces is characterized as ?self-eating?, whereby sequestration and intracelular breakdown of long-lived proteins and damaged organeles occurs during periods of celular stres (Mizushima and Klionsky, 2007). In Chapter 3, numerous autophagic structures (APSs) were observed throughout both the gastrodermis and epidermis of the anemone, Aiptasia pallida (Agasiz in Veril, 1864), after 12 h of heat stres treatment. These APSs contained sequestered host celular material and were often found within highly degraded regions of the cel that often exhibited sparse cytoplasm. Gastrodermal treatment tisues that exhibited elevated autophagic activity also displayed significant levels of symbiont loss. Thus, the authors suggested that autophagic activity occurred as an early response to heat stres conditions, and resulted in bleaching. Recent papers have highlighted the need for further investigations into the involvement of autophagy during breakdown of the cnidarian-dinoflagelate symbiosis (Davy et al., 2012; Weis, 2008). The aim of this study was to perform a quantitative examination of the abundance of APSs during heat stres in symbiotic and aposymbiotic, A.pallida tisues as wel as in the tisues of anemones treated with the known autophagy inducer, rapamycin. We have employed previously established methods used to initiate the autophagic response in Aiptasia pallida (Chapter 3). Quantification of autophagic activity in both symbiotic and aposymbiotic anemones 82 alowed us to draw conclusions about the probable role of each symbiotic member in the autophagic response. Materials and Methods Culture conditions Aiptasia pallida harboring symbionts typed as Clade A4 Symbiodinium (Santos et al. 2002; Scott Santos, ?personal communication?) were collected in the Florida Keys and maintained in artificial seawater (Ref Crystals) at 28-30 ppt salinity. Symbiotic anemones were kept in two 150 gal tanks at 24?1?C (such notation indicates mean ? standard deviation) below fluorescent light fixtures, each equipped with two 32W bulbs (Philips F32T8/TL841) producing 50?5 ?mol photons m -2 s -1 iradiance at the level of the anemones. Lights were set on a 12:12 h light/dark regime. A separate group of anemones, also collected from the Florida Keys, were rendered aposymbiotic by long-term (years) maintenance in complete darknes (except during brief additions of food) in a 150 gal tank at 24?1?C. The absence of Symbiodinium was verified by using PCR nuclear 18S rDNA (Rowan and Powers, 1991) and chloroplast 23S rDNA (Santos et al, 2002). In both cases no PCR product was produced (Scott Santos, ?personal communication?). Al anemones were fed freshly hatched Artemia larvae thre times per wek. Temperature-light treatments for both symbiotic and aposymbiotic anemones In order to ases whether or not elevated autophagy increases during heat stres-induced bleaching, two separate experiments were caried out. In a previously conducted experiment (Exp 1), symbiotic (n=3) anemones were exposed to heat stres conditions described in Chapter 83 3 known to cause non-lethal bleaching in A. pallida. In the second experiment (Exp 2), which is the present study and the focus of this investigation, both symbiotic (n=3) and aposymbiotic (n=3) anemones were exposed to the same heat-stres conditions as in Exp 1. In addition, controls were simultaneously run wherein symbiotic anemones were exposed to the same conditions as the stresed anemones but without the heat stres. In preparation for Exp 2, six symbiotic and six aposymbiotic medium sized anemones were placed in identical, individual plexiglas dishes that each contained ~200 ml of 0.45 ?m Milipore filtered artificial aquarium sea water (MFAW) and alowed to aclimate for ~5 days at the ambient lab temperature of 24?1 o C with 50?5 ?mol photons m -2 s -1 ambient light intensity. Water in these dishes was changed daily. Anemones were fed once during the first 3 days and then held unfed for 48 h prior to the beginning of the experiments. Both symbiotic and aposymbiotic treatment anemones were then transfered in their containers to an incubator at 32.5?0.5 o C and placed beneath two lamps each equipped with two 20W fluorescent bulbs (GE Ecolux F20T12/G50-ECO) that emited ~140?5 ?mol photons m -2 s -1 light intensity at the level of the anemones. The anemones were held in the incubator for 12 h and then removed to a dark, wel-ventilated box on the lab bench for 12 h in darknes at 24?1 o C. The incubator and dark box treatments were then repeated over the following 24 h for a total treatment time of 48 h. It took approximately 2.5 h for the 24?1 o C dishes to warm to 32.5?0.5 o C in the incubator under these conditions. This study primarily focuses on the efects of heat stres. There was a smal diference in light intensity betwen the anemone culture tanks (50?5 ?mol photons m -2 s -1 ) and the incubator (140?5 ?mol photons m -2 s -1 ); however, this is wel within the field light intensities (0 to > 1000 ?mol photons m -2 s -1 ) that these symbiotic anemones experience and was not considered by itself to be a significant stresor in our experiments. Of the six symbiotic and six 84 aposymbiotic anemones exposed to heat stres, tentacles from thre of each were sampled for fixation, embedding and examination with a transmision electron microscope (TEM) at t=0 h and t=48 h. Six additional symbiotic anemones and six additional aposymbiotic anemomes were aclimated as described above, and used as controls (no stres treatment) in this experiment. These were placed in the same type of dishes used for stresed anemones at 24?1?C with 50?5 ?mol photons m -2 s -1 iradiance for 12 h daily folowed by 12 h in darknes at 24?1?C over a 48 h period. In al cases water was changed daily and anemones were not fed during the 48 h treatment period. As was the case for the heat stresed anemones, tentacles from thre symbiotic and thre aposymbiotic anemones were sampled for fixation, embedding and TEM examination at t=0 h and t=48 h. Biochemical induction of autophagy Twelve symbiotic anemones were aclimated to lab conditions as described above. Six were then exposed to treatment conditions of 25 ?M rapamycin (Sigma-Alrich) and 1% DMSO solution in MFAW for 12 h (Dunn et al., 2007), and the remaining six to control conditions of 1% DMSO in MFAW for 12 h. Both control and treatment anemones were maintained on a 12:12 h light:dark regime at 24?1?C as described above. Tentacles from thre rapamycin treated anemones and thre control anemones were sampled for fixation, embedding and TEM examination at t=12 h. 85 Fixation, embedment and sectioning for TEM Anemones from heat stres treatments, rapamycin treatment, and control groups were prepared for TEM following the protocol outlined in Chapter 3. Heat treatment and control anemones (symbiotic and aposymbiotic) were sampled at 0 and 48 h and symbiotic rapamycin treatment and control anemones were sampled after 12 h. Anemones were relaxed in high Mg/low Ca seawater (Audesirk and Audesirk 1980) followed by the addition of chlorotone- saturated seawater. A subset of tentacles (n=6) was clipped from each of 3 randomly selected anemones at each time point (stres treatments and controls) (Fig. 22A). Tentacles were imediately fixed using methods similar to the protocol of Caroll and Kempf (1994) in Milonig?s phosphate buffered, gluteraldehyde fixative solution followed by a secondary fixation in a NaHCO 3 buffered, OsO 4 solution. Tisues were dehydrated through an ethanol series, transfered through thre changes of propylene oxide, and infiltrated and embedded using EMbed 812 resin (Electron Microscopy Sciences). Embedded tentacles were sectioned (~60nm) using a diamond knife (Diatome) on a Reichert-Jung Ultracut E microtome. Al sections were stained with uranyl acetate and lead citrate and examined using a Zeis EM 10C 10CR transmision electron microscope (TEM). Quantification of autophagy In order to quantify autophagic activity in A. pallida, tentacles were analyzed in each sampled anemone from each treatment [heat stres and controls (symbiotic and aposymbiotic) or rapamycin treated and controls] (Fig. 22) using a quantification method similar to that of Swanlund et al. (2010). First, one tentacle from each anemone was sectioned at mid-length in transverse orientation to generate one TEM section. Second, the transverse sections were viewed 86 in the TEM at low magnification and divided into four quadrants to ensure that regions representing the entire circumference of the tentacle could be analyzed. Third, both the gastrodermis and epidermis were photographed at the same magnification of 3,150X within the center of each quadrant (Fig. 22B). Negatives of the electron micrographs were scanned at 1200 dpi and converted to positive images. APSs were identified acording to morphological characteristics previously used to ases autophagic response in symbiotic cnidarian tisues as described in Chapter 3. Of the various stages of autophagy that were observed in this study, only the denser, more easily identifiable stages (autophagolysosomes) were evaluated. In order to ensure consistency, each APS had to fulfil two main criteria: 1) must have overal iregular periphery (to help distinguish from primary lysosomes and lipid droplets), 2) must have heterogenous appearance and contain visible sequestered cytoplasmic material (to distinguish from lipid droplets). Fourth, each image was analyzed using ImagePro Plus 7.0 software and measurements were made for i) total celular area outlined in the micrograph and i) total area occupied by APSs (Fig. 22C) similar to Oberley et al. (2010). The area occupied by symbionts within their symbiosomes was also determined and subtracted from the total cytoplasmic area outlined giving the adjusted cytoplasmic area (= the total cytoplasmic area that could potentialy contain gastrodermaly derived APSs (Fig. 22C). The overal area-based abundance of APSs was calculated in stresed symbiotic and aposymbiotic anemones, controls and rapamycin-treated animals as a percentage of the adjusted cytoplasmic area in both gastrodermis and epidermis [% APSs = [(area occupied by APSs) / (adjusted cytoplasmic area)] X 100]. 87 Figure 22: Quantification of autophagic structures within A. palida tentacle tisues. A) Tentacles were excised from live anemones (treatment and controls) and prepared for TEM. B) Transverse tentacle sections were viewed at low magnification on the TEM and photographs of both the gastrodermis (Ga) and epidermis (Ep) were taken within each of four quadrants (i-iv). Note: A light micrograph is used here for ilustrative purposes. C) Micrographs were then analyzed using ImagePro Plus 7.0 software to determine i) total cytoplasmic area, i) total area occupied by APSs, as well as ii) total area occupied by symbionts (Sy) for calculation of % APSs. Ga=gastrodermis, Ep=epidermis, gvc=gastrovascular cavity, Mg=mesoglea, Sy=Symbiodinium. Number quantities next to APSs signify area occupied by each. Number inside doted lines (surrounding symbionts) signifies the total area that they occupy. Scale bars = 60 ?m (B) and 5?m (C). Statistical analysis Diferences in % APSs were tested betwen 0 and 48 h for both stresed and control groups of symbiotic and aposymbiotic anemones and betwen rapamycin treated anemones and controls after 12 h using paired t-tests. Diferences in % APSs were also tested betwen gastrodermis and epidermis in treatment tisues after 48 h (symbiotic and aposymbiotic) or 12 hr (rapamycin). Comparison of the % APSs 88 A) betwen Exp 2 controls at t=0 h and Exp 2 controls at t=48 h, B) betwen Exp 1 t=0 h (pre-stres) and control t=0 h for Exp2 and C) betwen Exp 1 t=48 h of heat stres and Exp 2 t=48 h of heat stres showed no statistical diferences (p >0.277), so the stres treatment results for these two experiments were pooled for further statistical analyses. Since values were in the form of percentages, they were arcsin transformed for statistical analysis; however, results are presented as percentage values for greater clarity. Significance of the diference in average number of expeled Symbiodinium cels/anemone after 48 h betwen unstresed control and heat stresed treatment anemones was determined using a t-test. Al statistical tests were conducted using a significance level of 5% (alpha=0.05) with SAS 9.2 software. Algal cel counts In order to quantify symbiont loss resulting from heat-induced bleaching, expeled Symbiodinium cels were collected from both control and stresed anemone containers (n=3) after 48 h of treatment. For each anemone the collected algae were centrifuged, and then re- suspended in 250 mL of 0.45 ?m MFAW. Cels were then counted with a Neubauer hemacytometer (Perez and Weis, 2006) and the average number of algal cels that were expeled per anemone after 48 h was calculated for controls and heat stres treatment anemones. 89 Results and Discusion Autophagy in unstresed and heat-stresed symbiotic anemones Examination of unstresed (control) symbiotic A. pallida gastrodermis (Fig. 23A) and epidermis (Fig. 23D) revealed a consistently healthy celular appearance, which consists of intact epithelial cels with abundant cytoplasm (Chapter 3); however, as previously reported in Chapter 3, symbiotic tisues exposed to a 48 h heat-stres treatment exhibited numerous APSs within both host gastrodermal (Fig. 23B and C) and epidermal (Fig. 23E and F) cels. Large, dense APSs were commonly observed within degraded celular regions that exhibited markedly sparse cytoplasm (Fig. 23F). Measurements of % APSs (= [(area occupied by APSs) / (adjusted cytoplasmic area)] X 100]) ranged from a low of 0.711% to a high of 7.511%. Values are given as the mean % area occupied by APSs ? S.D. In symbiotic anemones there were no significant diferences in unstresed control gastrodermal tisues (p=0.777) betwen 0 h (x? = 0.711%?0.360, n=3) and 48 h (x? = 0.744%?0.168, n=3) or in unstresed control epidermal tisues (p=0.277) betwen 0 h (x? = 0.609%?0.341, n=3) and 48 h (x? = 0.868%?0.150, n=3) (Fig. 24). In addition, no significant diferences were found betwen unstresed control gastrodermal tisues (p=0.304) at 0 h (x? = 0.711%?0.360, n=3) as compared to treatment tisues prior to heat stres (x? = 0.843%?0.192, n=6). Similarly, no diferences were found in unstresed control epidermal tisues (p=0.582) at 0 h (x? = 0.609%?0.341, n=3) as compared to treatment epidermal tisues prior to heat stres (x? = 0.576%?0.199, n=6). A statisticaly significant increase in % APSs was measured in both treatment gastrodermal tisues (x? = 7.551%?2.741, n=6, p=0.002) and in treatment epidermal tisues (x? = 5.303%?3.411, n=6, p=0.004) betwen 0 h and 48 h of heat stres (Fig. 24). Numericaly, there was a slight diference in the means betwen the 90 gastrodermis (x? = 7.551%?2.741, n=6) and epidermis (x? = 5.303%?3.411, n=6) after 48 h of heat stres; however, that diference was not statisticaly significant (p=0.201) (Fig. 24). An overal lightening in the coloration of the treatment anemones was observed throughout the 48 h stres period indicating that the heat stres treatment induced a bleaching response. After 48 h a significant 12-fold increase (p < 0.0001) in expeled algae was observed in heat-treated anemones (x? = 827,213?4946, n=3) as compared to unstresed controls (x? = 68,880?902.1, n=3). These results indicate that autophagic activity does increase in both symbiotic (gastrodermal) and non-symbiotic (epidermal) tisue layers during heat stres induced-bleaching, but that no other external environmental efects play a major role in the response. Cel apoptosis (as determined by the criteria of Dunn et al. (2002; 2004) and in situ degradation of symbionts as described by Taylor (1973) and Brown et al. (1995) was rarely observed in host and/or symbiont cels after the heat stres treatment. As expected, symbiont degradation was rare in control animals. 91 Figure 23: TEM. Transverse sections of symbiotic Aiptasia palida tentacles at mid-tentacle length showing autophagic structures (APSs) in the gastrodermis (A-C) and epidermis (D-F) folowing heat stress. A) Symbiotic gastrodermal tisues at 0 h (no stres) treatment reveal a healthy ultrastructural appearance (note numerous symbionts = Sy), with few to no visible APSs in the host cytoplasm. B) Host tisues after exposure to 48 h of heat stress display numerous, dense, APSs and associated degraded regions, C) High magnification of boxed region in B showing dense APSs (asterisks) and degraded regions of cytoplasm (double black arrows), D) Epidermal tisue from a symbotic anemone at 0 h (no stress) treatment reveals a healthy appearance and few APSs, E) Host epidermal tisue after 48 h of heat stress treatment showing numerous APSs, F) Higher magnification of boxed region in E showing dense APSs (asterisks) surrounded by degraded cytoplasmic regions (double arrows). Scale bars = 2?m. 92 Figure 24: Percent autophagic structures within areas of gastrodermis and epidermis from symbiotic control anemones and within the gastrodermis and epidermis of treatment anemones after 0 and 48 h of control treatment or heat stress. Statistical significance was found in the change in % APSs present in both gastrodermis (p=0.02) and epidermis (p=0.04) of heat stress treatment anemones - designated with asterisks (paired t-test). The error bars represent standard deviation of the mean. Autophagy in unstresed and heat-stresed aposymbiotic anemones Unstresed aposymbiotic anemone tisues exhibited a normal overal appearance consisting of what might be described as a low iregular epithelium that was much thinner that that observed in symbiotic anemones. Unstresed aposymbiotic gastrodermal cels exhibited dense cytoplasm that contained few to no degraded regions or visible APSs (Fig. 25A). Alternatively, APSs and highly degraded celular regions were observed in aposymbiotic gastrodermal tisues subjected to 48 h heat stres treatment (Fig. 25B and C) similar to those of symbiotic tisues (Fig. 23B and C). Measurements of % APSs in aposymbiotic anemones revealed that there were no significant diferences in unstresed control gastrodermal tisues (p=0.729) betwen 0 h (x? = 1.514%?0.432, n=3) and 48 h (x? =1.301%?0.613, n=3) or in unstresed control epidermal tisues 93 (p=0.291) betwen 0 h (x? =0.205 %?0.091, n=3) and 48 h (x? =0.320%?0.247, n=3). No significant diferences were found betwen unstresed control gastrodermal tisues (p=0.255) at 0 h (x?= 1.514 %?0.432, n=3) and treatment gastrodermal tisues prior to heat stres (x?= 1.15 %?0.079, n=3). Similarly, no diferences were found in unstresed control epidermal tisues (p=0.194) (x?=0.205%?0.091, n=3) and treatment epidermal tisues prior to or following heat stres (x?=1.611%?1.099, n=3) (Fig. 26). A statisticaly significant increase in % APS was sen in aposymbiotic treatment gastrodermal tisues after 48 h of heat stres (x?= 5.946%?0.584, n=3, p=0.004) (Fig. 26). A significant diference in % APSs was found betwen the gastrodermis (x?= 5.946%?0.584, n=3) and epidermis (x?=1.611%?1.099, n=3) after 48 h of heat stres (p=0.016) (Fig. 26). Stresed aposymbiotic tisues displayed increased numbers of APSs and degraded regions in the gastrodermis similar to that observed in stresed symbotic anemones. This observation supports our hypothesis that the autophagic response is primarily host-derived. Biochemical induction of autophagy In order to confirm the role of autophagy in APS formation and subsequent celular degradation during heat stres, several symbiotic anemones were exposed to 25?M rapamycin, a known inducer of autophagy (Noda and Ohsumi, 1998), in 1% DMSO for 12 h and % APSs was quantified as above. As noted in Chapter 3, examination of tentacle tisues from rapamycin treated anemones revealed a strikingly similar celular appearance to those exposed to heat stres, exhibiting numerous APSs and degraded regions of cytoplasm (Fig. 25E and F). 94 Figure 25: TEM: Transverse sections of Aiptasia palida tentacles at midlength. Autophagic structures (APSs) form in aposymbiotic anemones (A-C) folowing heat stress and in rapamycin treated symbiotic anemones (D-F). A) Aposymbiotic gastrodermis tisues at 0 h (no stress) treatment reveal healthy ultrastructural appearance with few to no visible autophagic structures. B) Gastrodermal tisues at 48 h (stress treatment) display APSs and associated degraded regions (note degraded nematocyst in gastrodermis), C) Higher magnification of a dense APS (asterisk) D) Control symbiotic gastrodermal tisues exposed to 1% DMSO at 12 h (no stress) exhibit litle to no APSs with no cytoplasmic degradation, E) Symbiotic gastrodermal tisues exposed to 12 h of 25 ?M rapamycin result in the presence of numerous dark APSs with surrounding degraded cytoplasm, and F) Higher magnification of dense APSs (asterisks) in rapamycin treated gastrodermis. Scale bars = 2?m. 95 Figure 26: Percent autophagic structures within the gastrodermis and epidermis of aposymbiotic control and treatment anemones after 0 and 48 h. Statistical significance was found in the change in % APSs present in the gastrodermis of treatment anemones - designated with single black asterisk (paired t-test, p = 0.04). A significant difference in %APSs was also found between the gastrodermis and epidermis in treatment anemones after 48 h ?designated by double gray asterisks (t-test, p=0.016). The error bars represent standard deviation of the mean. A statisticaly significant increase in % APS was measured in treatment gastrodermal tisues incubated in 25?M rapamycin in 1% DMSO (x? =9.134%?5.524, n=3, p=0.006) as compared to control tisues incubated in 1% DMSO for 12 h (x? = 1.778%?1.206, n=3) (Fig. 27). A statisticaly significant increase in the % APS was also measured in treatment epidermal tisues incubated in 25?M rapamycin in 1% DMSO (x? =3.883%?1.694, n=3, p=0.005) as compared to control tisues incubated in 1% DMSO for 12 h (x? =1.245 %?0.333, n=3) (Fig. 27). A significant diference in % APSs was found betwen the gastrodermis (x? =9.134%?5.524, n=3) and epidermis (x? =3.883%?1.694, n=3) after 12 h of rapamycin treatment (p=0.010) (Fig. 27). Since rapamycin has previously been employed to induce autophagy in a variety of taxa (Ohsumi and Mizushima, 2004), including A. pallida (Dunn et al., 2007; Chapter 3), these results 96 provide further evidence that the structures identified here and in heat stresed tisues were autophagic in origin. Similar to what was observed for heat stresed symbiotic anemones, a visible layer of expeled algae was present on the bottom of each treatment anemone container after 12 h of exposure to rapamycin. Rapamycin-treated anemones also exhibited an overal lightening in the coloration of al anemones when compared to DMSO-incubated controls. This finding suggests that autophagy, whether induced biochemicaly or by heat stres, can initiate a bleaching response. Figure 27: Percent autophagic structures within the gastrodermis and epidermis of symbiotic control (12 h in 1% DMSO) and treatment (12 h in 25 ?M rapamycin + 1% DMSO) anemones. Statistical significance was found in the difference in % APSs present in both gastrodermis (p=0.06) and epidermis (p=0.05) of treatment anemones as compared to control anemones-indicated by single black asterisks (paired t-test). A significant difference in % APSs was also found between the gastrodermis and epidermis in treatment anemones after 12 h ?designated by double gray asterisks (t-test, p=0.010). The error bars represent standard deviation of the mean. 97 Conclusions Autophagy is a highly conserved response pathway that has been identified in a wide variety of taxa (Boya et al., 2005). The autophagic proces is tightly regulated by the inhibitory action of the protein kinase, target of rapamycin (TOR), which is controlled through the PI3K/AKT signaling pathway (Levine and Yuan, 2005; Lum et al., 2005). This autophagic pathway regulates a series of developmental stages (described in Chapter 3) that begins with the inclusion of celular structures/materials within a vacuole, followed by a fusion of that vacuole with a lysosome and degradation of the contents via hydrolytic enzymes. After degradation has occurred, the products are released into the cytoplasm to support biosynthesis of new proteins and other esential materials (Nivon et al., 2009). In this study, we have performed quantitative measurements on heat stresed symbiotic anemones that verify the autophagic response was widespread and significantly afected both the symbiotic gastrodermal and non-symbiotic epidermal layers. Based on these observations and those made in Chapter 3, we investigated the potential role of the symbionts during the autophagic response by exposing aposymbiotic anemones to an identical heat treatment as symbiotic anemones. Our results demonstrated that aposymbiotic gastrodermal tisues also exhibited a significant increase in autophagic degradative activity after 48 h of heat treatment. These findings indicate that the presence of symbionts is not necesary to initiate an autophagic response to heat stres and thus, that the up-regulation of autophagic activity in stresed anemones is primarily host-derived. While quantitative changes in autophagic activity were not significant in the epidermis in aposymbiotic heat-stresed anemones, such upregulation of autophagy did occur in the epidermis 98 of heat stresed symbiotic anemones. This suggests that the presence of symbionts may enhance the autophagic response in host anemones by contributing additional ROS that would increase oxidative stres levels throughout host tisues. Interestingly, the gastrodermis had significantly higher levels of autophagic activity than the epidermis in both the aposymbiotic and rapamycin- treated tisues. This result suggests that there may be physiological diferences betwen gastrodermal and epidermal tisue layers, such as sensitivity to oxidative stres. However, the fact that in heat stresed symbiotic anemones, both the gastrodermis and epidermis exhibited similar levels of autophagic activity suggests that the heat stres response may induce a diferent autophagic pathway than rapamycin. This variation in stres pathways may also acount for the decreased level of autophagic degradation observed in rapamycin treated tisues compared to those heat-stresed. Autophagy occurs at basal levels in most cels (Ohsumi and Mizushima, 2004) and functions to maintain cel homeostasis by degrading long-lived proteins and/or damaged organeles (Gozuacik and Kimchi, 2007; Levine and Yuan, 2005; Nivon et al. 2009). However, during stresful conditions, autophagy functions primarily to protect cels from significant injury (Swanlund et al., 2008). For example, it is thought that during periods of nutrient deprivation, autophagic activity is upregulated in order to promote cel survival by maintaining mitochondrial ATP energy production through the use of recycled nutrients (Levine and Yuan 2005). Hyperthermic stres conditions can also induce autophagy (Nivon et al., 2009; Oberley et al., 2008; Prasad et al., 2007; Swanlund et al., 2008) in order to clear the cel of damaged organeles and misfolded proteins (Nivon et al., 2009). In adition, autophagy may also function to eliminate cels altogether via autophagic cel death (Gozuacik and Kimchi, 2007; Kourtis and Tavernarakis 2009; Xue et al. 2001). 99 In this current study and in Chapter 3, heat stresed autophagic tisues contained numerous APSs and exhibited varying degres of celular degradation. Extensively degraded cels, where organeles and major portions of the cytoplasm were removed, exhibited characteristics of autophagic cel death (Gozuacik and Kimchi, 2007). These cels were frequently observed in both symbiotic and aposymbiotic heat stresed tisues, primarily in the gastrodermis. However, no similar celular degradation was observed in tisues after exposure to rapamycin, which is known to induce autophagic activity, but not autophagic cel death (Levine and Yuan, 2005). This suggests that the major cytoplasmic degradation observed in many cels of heat stresed symbiotic anemones was an indicator of autophagic cel death events and that this mechanism may be a major contributor to host tisue damage during bleaching events caused by heat stres. Heat stres may upregulate autophagy in an atempt to promote cel survival by recycling nutrients from digestion of damaged organeles and other cytoplasmic constituents. However, if the stres is severe or persists long enough, the damage resulting from autophagy may outweigh positive factors and autophagic cel death may lead to extensive and perhaps irecoverable tisue damage and eventual host death. The transition from pro-survival to pro-death pathways is commonly determined by the duration and intensity of the stres conditions (Gozuacik and Kimchi, 2007). Here we provide evidence to confirm that autophagic activity is upregulated during the first 48 h of sub-lethal temperature stres conditions in the absence of significant levels of other cel death mechanisms. It has been shown in other systems that autophagy can occur in the absence of apoptosis or necrosis in response to mild heat stres, but not acute heat shock (Komata et al., 2004). Thus, further investigations are necesary to determine whether it is possible that cel apoptosis and/or 100 necrosis may additionaly occur or replace autophagy after the 48 h stres period examined in our research. 101 V. Diferential gene expresion responses to elevated temperature in symbiotic Aiptasia pallida anemones using RNA-Seq Abstract Coral refs have dramaticaly declined over the past few decades as a result of mas mortality bleaching events. Bleaching is as a stres response to elevated temperature and/or light conditions resulting in the loss of intracelular dinoflagelates of the genus Symbiodinium from host gastrodermal tisues. This proces involves a complex series of events that occur throughout the duration of the bleaching episode and involve celular interactions betwen both symbiotic members. However, few studies have investigated the early host stres response when symbiotic breakdown is initiated. In this study, molecular techniques were employed to characterize the host response during the first 48 hours of heat and light stres in Aiptasia pallida. Both symbiotic and aposymbiotic anemones were exposed to stres conditions of ~32.5 o C at 140 ?mol photons m -2 s -1 iradiance for 12 hours daily followed by 12 hours of darknes at ambient temperature over a 48 h period. Diferential gene expresion was measured at 0, 3, and 48 hours post stres onset using an RNA-Seq procedure. Results from this investigation indicate that the gene expresion profile of A. pallida changes during early stages of bleaching, and several key proceses were identified that are involved in the host response, including stres response, protein degradation/synthesis, calcium homeostasis, cel-cel interactions, and vesicle traficking. This study provides a beter understanding of the genetic determinants of stres tolerance in a host anthozoan, and offers further insight into the celular proceses that underlie coral bleaching. 102 Introduction Coral refs represent one of the most productive and diverse ecosystems on earth; however, numerous anthropogenic stresors currently threaten their survival (Hoegh-Guldberg et al., 1999, Donner et al., 2007). Coral decline results primarily as a result of the symbiotic breakdown betwen corals and their intracelular photosynthetic dinoflagelates (=Symbiodinium) through a proces known as coral bleaching. Although coral bleaching can occur in response to a variety of environmental disturbances, elevated temperatures asociated with global climate change are one of is the primary cause of large-scale mas mortality bleaching events (Hughes, 2003; Hoegh-Guldberg et al., 2007). Symbiotic breakdown is initiated when high levels of reactive oxygen species (ROS) are produced during temperature or light stres (Leser, 1996; Franklin et al., 2004; Leser, 2006). ROS production has been shown to occur at high levels in thermaly stresed symbionts leading to decreased photosynthetic capability of the symbionts through photoinhibition and subsequent photodamage to photosystem I (PSI) (Warner et al. 1999; Leser 2006). Alternatively, host cels also independently produce ROS as a result of thermal stres-induced mitochondrial membrane damage (Ni and Muscatine, 1997; Dunn et al., 2012; Chapter 3). When ROS overwhelm the intracelular antioxidant defenses and directly damage celular structures, bleaching often results. Our current information surrounding the bleaching proces has historicaly been biased towards the response of the algal symbiont (Baird et al., 2008; Weis, 2008). Thus, our current understanding of the responses of corals or other symbiotic cnidarian hosts remains limited (Weis, 2008; Desalvo et al., 2011) despite increasing evidence that suggests that symbiotic dysfunction begins in the host cel (Ainsworth et al., 2008; Dunn et al., 2012; Chapter 3). 103 Recent advancements in molecular techniques, such as cDNA microarays and other genetic methods have provided valuable information on how thermal stres impacts a variety of coral hosts (Downs et al., 2000; Edge et al,. 2005; Morgan et al., 2005; Perez and Weis 2006; Richier et al., 2006, 2008; Foret et al., 2007; DeSalvo et al. 2008; Schwarz et al. 2008; Meyer et al., 2011). These studies enable us to addres important questions surrounding future bleaching scenarios and develop acurate models of the series of celular events that occur in the symbiotic host that result in collapse of the symbiosis (Weis, 2008). However, such investigations have been limited by the lack of an experimentaly tractable system. The anemone, Aiptasia pallida, provides a suitable model system to examine this problem (Weis et al., 2008; Sunagawa et al., 2010; Lehnart et al., 2012). Aiptasia maintains a symbiotic relationship with Symbiodinium spp. (Sunagawa et al., 2010), and the lack of a calcareous skeleton alows cel biological and microscopical manipulations to be conducted much more easily than in corals. Aiptasia also can grow very quickly and are very inexpensive and easy to maintain in the lab. Aiptasia spp. maintains a similar relationship with a variety of Symbiodinium spp. as do some corals, but can also exist in an aposymbiotic (symbiont-fre) state (Weis et al., 2008; Sunagawa et al., 2010; Lenhart et al., 2012). Investigations using the Aiptasia model can reveal key celular events that occur during various stages of the symbiosis, including establishment, maintenance, and breakdown. More recently, significant progres has been made in sequencing technology that has made possible the sequencing and de novo asembly of whole transcriptomes from non- model organisms (Meyer et al., 2009, 2011; Lehnart et al., 2012). Transcriptomic and EST sequences have been made available from a variety of corals (Schwarz et al., 2008; Meyer et al., 2009; 2011; Voolstra et al., 2009) as wel as a number of symbiotic anemones, including A. pallida (Sunagawa et al., 2009; Lehnart et al., 2012), Anemonia viridis (Sabourault et al., 2009), 104 and Anthopleura elegantisima (Richier et al., 2008). These sequence data alow comparative genomics to be asesed among the Scleractinia or Anthozoa, respectively. Furthermore, several cnidarian genomes have recently been completed, and include Nematostela vectensis (Putnam et al., 2007), Hydra magnipapilata (Chapman et al., 2010), and Acropora milepora (Shinzato et al., 2011), enabling genetic analyses to be conducted within members of the phylum. In addition, the transcriptomes of Symbiodinium spp. clades A and B have been made public (Bayer et al., 2012), which wil alow algal sequences to be distinguished from those of the host. This study used RNA-Seq to characterize how the gene expresion profile of A. pallida changes during early stages of bleaching. Several key functional pathways are identified that are involved in the host stres response. In particular, changes in the expresion of genes with roles in the stres response, protein degradation/synthesis, calcium homeostasis, cel-cel interaction, and vesicle traficking were of the most abundant. This study provides a beter understanding of the genetic determinants of stres tolerance in a host anthozoan, and offers further insight into the celular proceses that underlie coral bleaching. Materials and Methods Culture conditions Aiptasia pallida harboring symbionts typed as Clade A4 Symbiodinium (Santos et al. 2002; Scott Santos, ?personal communication?) were collected in the Florida Keys and maintained in artificial seawater (Ref Crystals) at 28-30 ppt salinity. Symbiotic anemones were kept in two 150 gal tanks at 24?C below fluorescent lamps producing 50?5 (? standard deviation) ?mol photons m -2 s -1 iradiance at the level of the anemones. Lights were set on a 12:12 h light/dark regime. 105 A separate group of anemones, also collected from the Florida Keys, were rendered aposymbiotic by long-term (> 10 years) maintenance in complete darknes in a 150 gal tank at 24?C. The absence of Symbiodinium was verified by using PCR nuclear 18S rDNA (Rowan and Powers, 1991) and chloroplast 23S rDNA (Santos et al, 2002). In both cases no PCR product was produced (Scott Santos, ?personal communication?). Al anemones were fed freshly hatched Artemia larvae thre times per wek. Temperature treatments for symbiotic and aposymbiotic anemones Both symbiotic and aposymbiotic anemones were subjected to the following heat stres treatment previously described in Chapter 3. Both symbiotic (n=6) and aposymbiotic (n=1) medium sized anemones were placed in identical, individual dishes and alowed to aclimate for ~4 days at the ambient room temperature of 24 o C under fluorescent light fixtures, each equipped with two 32W bulbs (Philips F32T8/TL841) producing 50?5 ?mol photons m -2 s -1 iradiance at the level of the anemones. Lights were set on a 12:12 h light/dark regime. Water in these dishes was changed daily and the anemones were fed once during the first 3 days and then held unfed for 48 h prior to the beginning of the experiments. Both symbiotic and aposymbiotic treatment anemones were then transfered in their containers to an incubator at 32.5 o C and placed beneath two lamps each equipped with two 20W fluorescent bulbs (GE Ecolux F20T12/G50-ECO) that emited ~140 ?mol photons m -2 s -1 light intensity at the level of the anemones. The anemones were held in the incubator for 12 h and then removed to a dark, wel-ventilated box on the lab bench for 12 h in darknes at 24 o C. The incubator and dark box treatments were then repeated over the folowing 24 h for a total treatment time of 48 h. It took approximately 2.5 h for the 24 o C dishes to warm to 32.5 o C in the incubator under these conditions. This study primarily focuses 106 on the efects of heat stres. There was a smal diference in light intensity betwen the anemone culture tanks (50 ?mol photons m -2 s -1) and the incubator (140 ?mol photons m -2 s -1 ); however, this is wel within the field light intensities (0 to >1000 ?mol photons m -2 s -1 ) that these symbiotic anemones experience and was not considered to be the primary stresor in our experiments. After exposure to 0, 3, 12, 24, or 48 h of heat stres, approximately ? of the tentacle crown of symbiotic and whole tentacle crowns of aposymbiotic anemones were excised using forceps and iridectomy scisors and imediately transfered to RNAlater and stored at -20 o C. Preparation of cDNA for Ilumina sequencing Total RNA was extracted from each sample using 1 mL TRIzol and treated with DNAse I to remove residual genomic DNA contamination. cDNA tag libraries were prepared from each RNA sample (n=3 symbiotic and n=1 aposymbiotic for each sampling time) as previously described for the SOLiD System (Meyer et al, 2011), substituting adaptor sequences appropriate for the Ilumina HiSeq sequencing platform. Each library was labeled with a sample-specific 6- bp barcode and gel-extracted to isolate the 350-500 bp size fraction. Sequencing, filtering and mapping cDNA libraries were pooled for sequencing on a single lane of Ilumina HiSeq 2000 (100-bp read length) at Oregon State University?s Center for Genome Research and Biocomputing (CGRB). Libraries were initialy prepared using custom adaptors that incorporated barcodes (?indices?) on both the 5? and 3? end of the construct, so that sample identities were encoded by unique combination of barcodes. Samples were pooled for multiplex sequencing in a single lane on the Ilumina HiSeq 2000 (100-bp SE reads) using standard Ilumina reagents to sequence the insert and the 3? barcode (?index 1?), but with a custom 107 sequencing primer for the 5? barcode (?index 2?). Because we found low sequencing eficiency with this custom primer, which prevented demultiplexing of samples based on barcode sequences, we subsequently prepared additional libraries for the 0, 3, and 48 hr samples using new adaptors compatible with standard Ilumina TruSeq primers. These new libraries were pooled for multiplex sequencing in a single lane of Ilumina HiSeq 2000 SE 100-bp reads at CGRB. Sequences were procesed prior to mapping to remove low-quality and uninformative reads. First, the four non-template bases introduced by reverse transcriptase were trimed off the raw sequences at the 5? end of each read. Low-quality reads having more than 50 bases with quality scores below 20 were excluded using custom Perl scripts. Any high-quality (HQ) reads matching adaptors used in library preparation (?15 matching bases) were identified using cross_match (REF) and excluded using custom Perl scripts. Analysis of tag-based RNA-Seq data requires a reference asembly against which to map the reads, as previously described (Meyer et al, 2011). We initialy atempted to analyze these data using a recently published transcriptome asembly for A. palida (Lehnart et al., 2012), but found that redundancy in the asembly led to a substantial loss of data because of ambiguous matches. To addres this we constructed a custom transcriptome asembly using sequence data from the published transcriptome (Lehnart et al., 2012), but including only reads derived from aposymbiotic samples. Reads were first filtered to eliminate low-quality reads and reads matching adaptors, as described above. The HQ reads that remained were asembled using Trinity (Grabher et al, 2011) with minimum kmer coverage = 2. Asembled transcripts were annotated by comparison against UniProt (v2012_06) using BLASTX (e-value threshold = 10 -4 ). 108 Identification of diferentially expresed genes (DEGs) Al calculations and statistical tests were conducted using the R statistical software (version 3.0.0) (R Development Core Team, 2008). We focused our atention on transcripts expresed at ?2 reads per sample on average, and further excluded any transcripts present in symbiotic samples, but absent in aposymbiotic samples, which might be derived from Symbiodinium rather than Aiptasia. Read counts were summed across sequencing runs by sample. Expresion diferences were tested using a negative binomial test implemented in the R package DESeq (version 1.12.0) (Anders & Huber, 2010). To identify genes diferentialy expresed in response to the heat stres treatment, we compared expresion profiles at the early (3 hour) and late (48 hour) sampling points with initial samples (0 hours). The un-replicated aposymbiotic samples were not used for statistical comparisons, but only used to verify that transcripts were derived from host rather than symbiont. False discovery rate (FDR) was controlled at 5% (Benjamini & Hochberg, 1995). Results and Discusion Sequencing, mapping and assembly Altogether, 115,131,741 raw reads were obtained by sequencing pooled libraries. 82,223,991 HQ reads (71%) remained after quality and adaptor filtering. The gene from which each read originated was identified by mapping HQ reads against an annotated aposymbiotic Aiptasia transcriptome. 61,737,036 of these (75% of HQ reads, or 54% of raw reads) were aligned unambiguously against the reference transcriptome. 109 Response to temperature treatments Statistical analysis of RNA-Seq counts data (the number of reads mapped to each gene in each sample) revealed large shifts in gene expresion resulting from elevated temperature stres. Exposure to 3 h of heat stres induced the diferential expresion of 293 genes in symbiotic anemones (negative binomial model, FDR = 0.05) (Fig. 28). Of these, 213 genes were upregulated and 80 were downregulated. In later samples (48 h) only 21 genes were diferentialy expresed, of which 6 were upregulated and 15 were downregulated (Fig. 29). Fold changes ranged from +30.62 to -9.90. 232 of the 293 DEGs identified in 3 h samples (79%) matched known proteins (based on a BLASTX search against UniProt), and 7 of the 21 genes identified in the 48 h timepoint. 110 Figure 28. Cluster analysis of differentially expressed genes from control anemones (0A-C, n=3) and anemones exposed to 3 h of thermal stress treatment (3A-C, n=3). Top dendrogram shows distinct clustering of treatment anemones (3A-C) relative to control anemones (0A-C). Side dendrogram shows clustering of genes based on type. Colors represent gene expression patterns; yellow indicates upregulated genes and blue indicates downregulated genes (see color/expression scale in uper left corner). 111 Figure 29. Cluster analysis of differentially expressed genes from control anemones (0A-C, n=3) and anemones exposed to 48 h of thermal stress treatment (48A-B, n=2). Top dendrogram shows distinct clustering of treatment anemones (48A-B) relative to control anemones (0A-C). Side dendrogram shows clustering of genes based on type. Colors represent gene expression patterns; yellow indicates upregulated genes and blue indicates downregulated genes (see color/expression scale in uper left corner). A subset of the diferentialy expresed genes (DEGs) produced in this study were categorized into putative functional groups acording to GO molecular function or manualy defined functions based on literature and database searches (Table 1). After 3 h of thermal stres, the most abundant DEGs were those that had putative functions in the i) general stres response, i) protein/endosome sorting-asembly, ii) protein degradation, iv) calcium binding/transport, and v) cel adhesion. A majority of these DEGs were upregulated. After 48 h of thermal stres, majority of the annotated DEGs had putative functions involved in i) protein degradation, i) cel adhesion, and a wide variety of ii) miscelaneous proceses that were mostly downregulated. In 112 both the 3 and 48 h samples many of the DEGs could not be identified based on sequence comparisons (Table 1). 113 Table 1. Differentialy expresed genes after 3 h and 48 h of thermal stress treatment relative to 0 h controls categorized into functional groups. Gene/Function 1 Match 2 Sym 3h FD 3 P value 3a Sym 48h FD 4 P value 4a Stress response Heat shock protein 70 (HSP70) Q5FB18 7.58 <0.001 Heat shock protein 90 alpha (HSP90a) G4W8Y9 7.14 <0.001 Hypoxia upregulated protein 1 (hyou1) F1QUW4 5.85 <0.0001 Cysteine-rich with EGF-like domain protein 2 (creld2) Q7SXF6 5.35 <0.001 DnaJ homolog subfamily C member 3 (dnajc3) Q7ZWH5 4.64 <0.001 Glucose-regulated protein 94 (GRP94) A5LG7 4.29 <0.001 Stres-70 protein, mitochondrial (HSPA9) Q5R51 3.15 <0.001 Universal stres protein A-like protein (CLF13016) H2KVS5 -2.41 0.002 Protein degradation/synthesis KRR1 smal subunit procesome component (v1g71320) A7S7I3 5.71 <0.001 Cathepsin L (CATL) C1BJ28 4.42 <0.001 Ubiquitin (UBIQ) C1BNT5 3.36 <0.001 Kelch-like protein 20 (KLHL20) F1NM8 2.58 <0.001 FAD-dependent oxidoreductase domain-containing protein 2 (FOXRED2) Q8IWF2 -3.75 0.018 Cation- dependent manose- 6- phosphate receptor (MPRD) B5X109 -3.45 0.002 -4.04 0.054 Calcium homeostasis Calumenin (CALU) Q5RDD8 3.63 <0.001 Sarco/endoplasmic reticulum calcium ATPase isoform B (SERCA) B2KR1 3.08 <0.001 Cel-cel interactions Galectin-3-binding protein (GW710920) G5AVF6 2.0 0.053 2.93 0.0237 Putative cel adhesion protein (Sym32) Q9NH96 2.84 <0.001 Ankyrin repeat protein, putative (TVAG13720) A2FNS2 -3.4 0.054 Vesicle traficking RAB26 (rab26) A0JM47 1.97 <0.001 Vesicle-traficking protein (SEC2B) Q5ZJW4 1.82 0.0313 Miscelaneous Heme binding protein (MICPUN58704) C1E6L8 8.9 <0.001 Cryptochrome 1 (CRY1) A2I2P0 5.21 <0.001 Wu:fb63a08 protein (wu:fb63a08) A5D6R8 2.15 0.020 -3.32 0.0164 Hemaglutinin/amebocyte agregation factor (HAF) B5XEP9 -3.75 0.018 Krox protein (Krox) Q9NFK6 -2.04 0.052 -3.92 0.0212 Cytochrome P450 (cyp46a1) O97689 -2.56 0.0131 PAR domain protein 1 (Pdp1) B0LD02 -6.64 0.061 Unknown comp20613 30.62 <0.001 comp3713 5.1 <0.001 comp2717 3.03 <0.001 3.84 0.009 comp414 -7.7 <0.001 comp15191 -9.90 <0.001 1 Genes chosen based on expresion values or functional significance and organized into functional groups designated acording to Uniprot biological/molecular function or manualy defined functions based on literature and database searches. 2 Match based on Uniprot acesion numbers 3 Fold diference values for symbiotic samples after 3 h relative to 0 h controls 4 Fold diference values for symbiotic samples after 48 h relative to 0 h controls a p-value (negative binomial model (DESeq), False discovery rate= 0.05) al <0.05 114 Table 1. Differentially expressed genes after 3 h and 48 h of thermal stress relative to 0 h controls categorized into functional groups that correspond to heatmaps in Figures 28 and 29. Genes represent a subset of the total population of differentially expressed genes chosen based on their high expression values and their functional significance as detected by RNA-Seq. Expression values are shown as fold change for each time point. Putative functional roles are designated according to Uniprot molecular/biological function or manually defined functions based on literature and database searches. Hierarchical clustering was used to group DEGs with similar expresion paterns in the 3 h (Fig. 28) and 48 h (Fig. 29) samples. For both time points, this revealed groups of genes upregulated and downregulated relative to the 0 hr controls. Samples showing similar expresion paterns were grouped in the same way, revealing that DEG showed reproducible changes across samples. The majority of DEGs after 3 h were upregulated (73%) relative to 0 hr controls (Fig. 28). Some of these highly upregulated genes were asociated with stres response (HSP70, HSP90a, hyou1, creld2, dnajc3, GRP94), protein degradation/synthesis (CATL, v1g71320, UBIQ), calcium homeostasis (CALU), cel-cel interactions (Sym 32), and vesicle traficking (rab26), while others had miscelaneous (MICPUN58704, CRY1) and unknown functions (e.g. comp20613, with a fold change of 30.62). A smaler subset of DEGs were downregulated (27%) after 3 h of temperature stres (Fig. 28). Some of these downregulated genes were asociated with stres response (CLF113016) and others had miscelaneous (Pdp1, cyp46a1) and unknown functions (e.g. comp4114, with a fold change of -7.77). In contrast to gene expresion profiles in the 3 h samples, a majority (71%) of DEGs were downregulated after 48 h of temperature stres (Fig. 29). Some of these highly downregulated genes were asociated with protein degradation/synthesis (MPRD, FOXRED2), cel-cel interactions (TVAG137200), and others had miscelaneous (Krox, HAF, wu:fb63a08) and unknown functions (e.g. comp15191, with a fold change of -9.90). A smaler subset (29%) of DEGs were upregulated after 48 h of temperature stres (Fig. 115 29). Some highly upregulated genes were asociated with cel-cel interactions (GW710920) and others had unknown functions (comp33713, with a fold change of 5.11). Diferential expresion across time points Several genes exhibited diferential gene expresion after both 3 and 48 h of thermal stres, five of which showed significance (p <0.05) in both timepoints (Table 1). These included 1) MPRD (downregulated at 3 and 48h), 2) GW710920 (upregulated at 3 and 48 h), 3) Wu:fb63a08 protein (upregulated at 3 h and downregulated at 48 h), 4) Krox (downregulated at 3 and 48h), and 5) an unknown protein comp27177 (upregulated at 3 and 48h). Some genes exhibited constituitive expresion at both 3 and 48 h timepoints (Figs 28 and 29). Notable DEGs included Sym32 (upregulated 2.84 and 2.66 fold respectively) and rab26 (upregulated 1.97 and 2.22 fold respectively). In contrast, several genes showed dramatic change in their expresion over time, the majority of which were upregulated after 3 h and either remained unchanged or were downregulated after 48 h of thermal stres (Figs 28 and 29). DGEs involved in the putative stres response that exhibited this patern were HSP70, HSP90a, hyou1, and creld2, which increased 7.58, 7.14, 5.85, and 5.35 fold, respectively, after 3 h of thermal stres. Upregulation of the genes decreased to 1.09, 1.84, 1.27, 1.36 fold after 48 h. Following the same trend, several DEGs involved in protein degradation, including v1g71320 and CATL, exhibited fold changes of 5.71 and 4.42 fold, respectively, and these values both decreased to 1.73 and 1.82 fold after 48 h. Another notable gene, FOXRED2, was downregulated -3.85 fold after 3 h and upregulated 1.20 fold after 48 h. Some highly expresed miscelaneous DEGs were Heme-binding protein (MICPUN58704) (upregulated 8.99 fold after 3 h and downregulated - 1.72 fold after 48 of thermal stres) and Pdp1 (downregulated -6.64 fold after 3 h and 116 upregulated 1.62 fold after 48 h). Major functions involved during temperature stres Stres response The majority of the genes that were categorized into putative functional groups were asociated with the stres response (Table 1). Interestingly, almost al of the DEGs in this group were highly upregulated after 3 h, but remained stable or were downregulated in 48 h samples. This finding supports previous suggestions that the stres response can vary with the length of the thermal treatment in Aiptasia spp. (Dunn et al., 2002, 2004; Chapter 3). The genes involved in this response include the ubiquitous heat shock proteins (HSPs), such as HSP70, HSP90a, etc. (Bromage et al., 2009). These proteins act as molecular chaperones that asist in folding newly synthesized proteins in order to maintain tertiary structure (Buckley and Hofmann, 2002). During temperature or light stres conditions, this response maintains proper cel function (Baird et al., 2009). Not surprisingly, RNA-Seq results from this study indicated the upregulation in several HSP genes, including HSP70, HSP90a, dnajc3 (HSP40), and Stres-70 protein (HSPA9) in heat stresed A pallida after 3 h. These results agre with previous studies documenting the induction of HSPs in response to thermal stres in a variety of cnidarians (Sharp et al., 1994, 1997; Black et al 1995; Fang et al., 1997; Downs et al., 2000, 2002, 2005; Gates and Edmunds, 1999; DeSalvo et al., 2008; Meyer et al., 2011). Other DEGs asociated with ER-stres, often caused by mis/unfolded proteins in the lumen of the endoplasmic reticulum (Ron and Walter, 2007), were upregulated in this study after 3 h of thermal stres, which have been previously documented in cnidarians, including hypoxia 117 upregulated protein 1 (hyoup1) (Aranda et al., 2011) and glucose-related protein 94 (GRP94) (Sharp et al. 1994; Hashimoto et al., 2004). Protein degradation/synthesis Numerous genes asociated with a protein degradation or synthesis function were diferentialy expresed after 3 h of thermal stres (Table 1). The most highly upregulated gene in this functional group was v1g71320, which encodes the KR, a smal subunit procesome component protein. As the name implies, KR1 proteins appear to be esential components of rRNA maturation and ribosome biogenesis (Gromadka and Rytka, 2000). The remaining five DEGs were involved in the proces of protein degradation and included CATL, UBIQ, KLHL20, FOXRED2, and MPRD (Table 1). In our analysis, Cathepsin-L (CATL) was upregulated after 3 h. Cathepsins are lysosomal cysteine proteases that break down intracelular and endocytosed proteins, particularly those asociated with the extracelular matrix (Bromme and Wilson, 2011). Ubiquitin (UBIQ) is an important marker of protein degradation and stres in most phyla, including corals and other cnidarians (Downs 2000; Brown 2002; Fauth, 2006). Ubiquitin functions to tag stres-damaged proteins for degradation by proteolytic complexes termed ?proteasomes? (Hershko and Ciechanover, 1998). A measured increase in ubiquitin levels indicates higher levels of protein degradation and turnover (Gof et al., 1988). Kelch-like protein (KLHL20) was also upregulated only after 3 h of thermal stres. KLH20 plays a role in negatively regulating cel death activity by ubiquitinating and degrading death-asociated- protein-kinase (DAPK) (Le et al., 2010). In contrast, one gene involved with the putative protein degradation function, FAD- dependent oxidoreductase domain-containing protein 2 (FOXRED2) only showed downregulation after 48 h of thermal stres. FOXRED2 is an ER protein that regulates 118 proteasome activity and may decrease the stres response and cel death when downregulated (Shim et al., 2011). Cation-dependent mannose 6-phosphate receptor (MPRD) was significantly downregulated after both 3 and 48 h of thermal stres. MPRD is involved with lysosome degradative pathway and functions to target lysosomal enzymes to the lysosome (Sleat et al., 2006). This suggests that organele function may be impaired during thermal stres, which is a known consequence of ROS damage in corals (Richier et al., 2005). Calcium homeostasis Two DEGs asociated with calcium homeostasis, CALU and SERCA, were upregulated after 3 h of thermal stres (Table 1). Calumenin (CALU) is a Ca 2+ -binding protein and member of the CREC protein family and has been identified during the heat and/or light stres response in cnidarians (Aranda et al., 2011; Belantuano et al., 2012; Moya et al., 2012). Belantuano et al., (2012) found a decrease in expresion of calumenin in response to heat stres in corals that had not been pre-conditioned and an increase in pre-conditioned (thermaly-tolerant) corals after eight days of thermal stres. Furthermore, Ganot et al. (2009) identified calumenin as the most highly upregulated gene of the symbiotic condition in Anemonia viridis and suggested that calumenin may play a key role in the cnidarian/dinoflagelate symbiosis. Additionaly, these authors proposed that calumenin may be involved in host/dinoflagelate recognition mechanisms through regulation of the anthozoan cel adhesion protein, Sym32. The fact that CALU was upregulated during our heat stres study suggests that calcium homeostasis was being disrupted after early stages of thermal stres. This finding lends support to previous studies that have suggested that calcium homeostasis may play an important role in the cnidarian response to heat stres (Fang et al., 1997; Huang et al., 1998; Sandeman, 2006). 119 Cel-cel interactions Several genes asociated with cel-cel interactions were diferentialy expresed after varying lengths of thermal stres (Table 1). Galectin-3-binding protein, GW710920, was upregulated after 3 and 48 h of thermal stres. Galectin-3 is a carbohydrate-binding protein (Barondes et al., 1994) that has been shown to inhibit apoptosis through cysteine protease pathways (Akahani et al., 1997). Thus, the observed upregulation of GW710920 throughout the heat stres treatment in our analysis may suggest that apoptosis was being inhibited. This current study used the identical heat stres treatment outlined in Chapter 3, (with only a smal increase in iradiance from approximately 50 to 140 ?mol photons m -2 s -1 iradiance) with Aiptasia pallida, which resulted in low levels of host/dinoflagelate apoptosis in both experiments. Another gene, putative cel adhesion protein (sym32) was upregulated after 3 h of thermal stres. sym32 is a fasciclin domain-containing protein that was first described as a ?symbiosis gene? because it was shown that to be highly expresed in the symbiotic state in Anthopluera elegantisima (Reynolds et al., 2000). The authors concluded that the sym32 is involved in regulation of the symbiosis through mediating cel-cel interactions. In a separate investigation, this gene was found to localize to membranes that surround the symbiont within the host cels, further suggesting that it plays an important role in host/dinoflagelate interactions communication and signaling (Schwarz and Weis, 2002). Furthermore, heat with or without UVR stres has been shown to induce the downregulation of both sym32 and calumenin in A. viridis (Moya et al., 2012). Thus, the fact that sym32 and CALU were upregulated after 3 h of heat stres in our analysis further suggests that calumenin plays a role in regulating sym32. Additionaly, sym32 is likely involved in host-dinoflagelate interactions in Aiptasia, which are enhanced during early stages of thermal stres. However, the results of our analysis were in 120 contrast to those previously reported in A. viridis (Moya et al., 2012) since we found a significant upregulation of CALU and sym32 in response to heat stres. This observed upregulation may be due to enhanced host-dinoflagelate signaling resulting from the heat stres response. Vesicle traficking Two genes asociated with vesicle traficking, including rab26 and cyp46a, were upregulated after 3 h of thermal stres (Table 1). Ras-like smal GTPases, such as rab26, are involved in regulated vesicular secretion in eukaryotic cels (Wagner et al., 1995; Yoshie et al., 2000; Nashida et al., 2006). Previous studies have also shown that dinoflagelates may manipulate host Rab GTPases, preventing lysosomal fusion with host phagosomes that they reside within in order to initiate and maintain the symbiosis (Fit and Trench 1983; Chen et al. 2003; Chen et al. 2004; Chen et al. 2005). Although it is possible that rab26 may play a similar role in the cnidarian-dinoflagelate symbiosis, this has not yet been established. Additionaly, the vesicle-traficking protein (SEC22B) functions in traficking betwen the endoplasmic reticulum and Golgi apparatus (Hay et al., 1997; Liu and Barlowe 2002). It is likely that both rab26 and SEC22B were increased in response to thermal stres, since cels are known to play critical roles in secretion and intracelular transport. Many molecules regulated by this proces are known to be involved in the stres response (DeMaio, 2011). These molecules are wel positioned to be intercelular signaling molecules, and may act as reporters of stres for other cels and tisues (DeMaio, 2011). Other heat-stimulated, diferentialy regulated transcriptional activity Numerous genes asociated with miscelaneous functions were diferentialy expresed after varying lengths of thermal stres (Table 1). The most highly upregulated gene in this functional groupresponse was by heme-binding protein (MICPUN58704), which was 121 upregulated (8.99 fold) after 3 h of thermal stres. Hemes are prosthetic groups that coordinate redox interactions within multiple intracelular pathways such as in the in electron transport chain and transport of diatomic gases. Many are required for oxygen storage and transport and therefore respiration. They are critical for the support of biosynthetic pathways for almost every form of life (Igarashi and Sun, 2006). Heme production is known to increase in conjunction with elevated xenobiotic detox pathways in cnidarians (Kramarsky-Winter et al., 2009). More recently, heme-binding proteins were shown to be upregulated in response to thermaly stresed corals, which may have resulted from iron-induced oxidative injury (Belantuono et al., 2012). Another DEG found in our analysis was cyp46a1, which was downregulated after 3 h of thermal stres. Cholesterol 24-hydroxylase, cyp46a1, is a member of the superfamily of heme- thiolate enzymes (Omura, 2005) that catalyze oxidative transformation leading to activation or inactivation of endogenous and exogenous substrates (Goldstone et al., 2010). cyp46a1 has been implicated as a critical gene for cholesterol homeostasis and membrane function in human neurons (Russel et al., 2009). Conclusions Here we have asembled a reference transcriptome for adult symbiotic Aiptasia palida using an Ilumina sequencing platform and characterized major celular proceses that occur as a consequence of thermal stres. Our findings suggest that exposure to a sub-lethal thermal treatment induces diferential expresion of a variety of celular activities, including stres response, protein degradation/synthesis, calcium homeostasis, cel-cel interaction, and vesicular traficking (Table 1). The majority of the genes related to the heat stres response were upregulated after 3 h of thermal treatment and remained stable or downregulated after 48 h of 122 treatment. Almost al the genes grouped within the stres response functional group folowed this trend, as did many genes involved with protein degradation, calcium homeostasis, and vesicle traficking (Table 1). Previous studies on corals have shown that these proceses are afected during thermal stres (Black et al., 1995; Fang et al., 1997; Sharp et al., 1997; Edge et al., 2005; DeSalvo et al., 2008). In the past 100 years sea surface temperatures have elevated by almost 1 o C and are currently increasing at the rate of approximately 1-2 o C per century (Hoegh-Guldberg, 1999). Thus, future studies would benefit by evaluating the potential of these diferentialy expresed genes to serve as potential biomarkers for cnidarian health. In doing so, we may be able to elucidate the celular and molecular mechanisms that underlie the heat-induced bleaching proces that threatens coral ref ecosystems worldwide. 123 Literature Cited Abraham, M. C. and S. Shaham. Death without caspases, caspases without death. Trends In Cel Biology. 14(4):184-193. Ainsworth, T. D., Hoegh-Guldberg, O., Heron, S. F., Skirving, W. J., and W. Leggat. 2008. 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The collagens of Hydra provide insight into the evolution of metazoan extracelular matrices. Journal of Biological Chemistry. 282:6792?6802. Zoccola, D., Tambutt?, E., Kulhanek, E., Puverel, S., Scimeca, J. C., Alemand, D., & Tambutt?, S. 2004. Molecular cloning and localization of a PMCA P-type calcium ATPase from the coral Stylophora pistilata. Biochimica et Biophysica Acta (BA)-Biomembranes. 1663(1):117-126. 142 General Conclusions Mas mortality bleaching events have significantly increased over the last 30 years (1993, 1998, 2005) resulting in a significant decline of coral cover and diversity in many regions of the world. N.O.A.. (2012) reported that ~90% of Caribbean corals experienced severe bleaching during the 2005 bleaching event, and only ~45% have since recovered. Similar mas mortality episodes are predicted to increase as global climate change results in more frequent high STs, endangering the future survival of coral refs worldwide. Increasing prevalence of bleaching events result primarily from steadily rising STs, which represent only one of the major efects of anthropogenic global climate change (N.O.A.., 2012). Other anthropogenic factors, such as pollution and overharvesting, may significantly exacerbate the weakened condition of corals during warmer seasons and have been linked to numerous disease outbreaks and bleaching events (N.O.A.., 2012). Although coral bleaching has been studied for the past few decades, there remains a great deal of ?big picture? concepts that currently remain unresolved. In particular, we know very litle of how corals imediately respond to elevated STs and much speculation remains regarding how symbionts are lost and whether the host and/or symbiont controls the bleaching proces (Weis, 2008). This study provides evidence that elevated autophagic activity results in degradation of host cels and contributes to the eventual loss of the symbiont through a novel celular bleaching mechanism, ?apical detachment? in the model symbiotic anemone, Aiptasia pallida. Results from 143 this investigation also demonstrated that the anthozoan host is the first member to respond to elevated temperature stres by regulating both host cel and symbiont abundance through autophagy. In addition, ultrastructural observations of a complex entanglement at the mesogleal- gastrodermal cel interface suggest that previous descriptions of the celular bleaching mechanism termed ?host cel detachment? is an unlikely mechanism for bleaching in Aiptasia and is in need of revision. Results from the RNA-Seq analysis revealed the highest levels of diferential gene expresion in symbiotic A. pallida anemones during early stages of a thermal stres treatment, which corroborated our ultrastructural findings. Several key genes and celular proceses were identified that provides a beter understanding of the genetic determinants of stres tolerance in a host anthozoan. These findings contribute esential information to our current level of understanding surrounding the bleaching proces and wil facilitate a beter understanding of how the global climate change wil afect coral health.