IDENTIFICATION OF LOCAL ADAPTATION IN PORITES ASTREOIDES INHABITING THE FLORIDA REEF TRACT: BIOTIC STRESS, A DISREGARDED FORCE OF CHANGE AFFECTING CORAL REEFS By Joshua A Haslun A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of Zoology - Doctor of Philosophy Ecology, Evolutionary Biology and Behavior - Dual Major 2015 ABSTRACT IDENTIFICATION OF LOCAL ADAPTATION IN PORITES ASTREOIDES INHABITING THE FLORIDA REEF TRACT: BIOTIC STRESS, A DISREGARDED FORCE OF CHANGE AFFECTING CORAL REEFS By Joshua A Haslun Up to 98 % of the total carbon acquired by scleractinian anthozoa n s (coral) is provided by carbon fixation from an algal symbiont ( Symbiodinium spp. ) . In return, the host provides protection, a source of nitrogen, and CO 2 for carbon fixation to the symbiont. The obligate nature of this symbiosis places constraints on the environmental conditions capable of sustaining growth, reproduction, and reef expansion. Although many environmental factors can be detrimental to corals c limate warming and disease have drastically altered coral communities over the past 40 years . C oral cover along the Florida Reef Tract (FRT) has decreased by 70 % and as a result the benthic reef community has transitioned from a coral dominated to a macro - algal dominated ecosystem. The primary contributors to the observed phase shift are disease and climate change. Increasing our understanding of coral resistance and sensitivity to disease and climate change related stressors is paramount when the future of this ecosystem is considered. Differential habitation of the inshore patch r eef system and offshore bank reef system is identi fied with a multivariate statistical approach among three coral species, Porites astreoides , Montastraea cavernosa , and Siderastrea siderea . All three species were observed to have increased size and abund ance at inshore reefs des pite the characterization of insh ore patch reefs as a habitat with increased levels of thermal stress an d eutrophication compared to offshore bank reef habitats . These results point to disease as a contributing factor affecting t he offs hore bank reef system. B ased on the extensive history of disease associate d with the offshore bank reef system, I hypothesized that offshore coral communities increase immune expression to deter disease . Because corals utilize an innate immune sys tem to detect and respond to ha rmful microorganisms , I applied quantitative reverse transcription real - time PCR of genes associated with immunity to estimate the degree of this response. Reciprocal transplantation of P. astreoides collected from a represe ntative inshore and offshore reef enabled the distinction between environmen tal and popula tion dependent effects. U pregulation of TNF receptor associated factor 3, a protein critical to bacterial and vi ral responses, was identified in corals experiencing the offshore habitat during the summer indicating increased d isease related stress during warmer period s . Corals originating offshore upregulated expression of a denylate cyclase associated protein 2 compared to inshore corals indicating an adaptive response to increased disease related stress . Population - dependent adaptive responses to temperature and pathogen related stress in P. astreoides were confirmed by challenging fragments originating from each site (n = 6) for 8 h with a contro l (28°C), increased temperature (32°C) , or a treatment of increase d seawater temperature and 5 µg ml - 1 lipopolysaccharide . Offshore coral fragments exposed to the synergistic treatment and inshore coral fragments exposed to increased SWT displayed r espons ive upregulation of genes . Responsive upregulation of genes was associated with increased variation in SWT for inshore corals and increased variation in pathogen associated molecular patterns response in offshore corals . The observed increase in express ion of the immune system by the offshore population was also associated with decreased coral abundance (survival) and decreased colony size, which in P. astreoides contributes to decreased fecundity. Therefore, survival of offshore corals in the face of p athogenic organisms likely comes at costs to other measures of fitness whereas inshore corals do not experience these pathogen related costs. Therefore, t wo populations of P. astreoides inhabit the FRT. As SWTs continue to increase from carbon emissions, thermal stress will likely exacerbate the effects of pathogen related stress impacting the offshore population and SWT stress will further impact the inshore population. Both populations are in peril but we identify pathogen associated stress as a concea led stressor. iv ACKNOWLEDGMENTS A doctoral education has been a lesson in perseverance , adap tation, and imagination. This experience has extended across state and country boundaries and because of this I was afforded the opportunity to learn from a wonderfully diverse group of individuals that deserve recognition beyond what I can hope to provide here . I would like to first thank my famil y for their c ontinued support during struggles and success es despite the distance between us. Secondly, I would like to thank Dr. Kevin Strychar for providing the opportunity to study a remarkable organism and ecosystem as well as the freedom to develop a project of my own design . I hope that this experience has been as rewarding for you as it has been for me. Third, I would like to thank both Nathaniel and Peggy Ostrom for accepting me into their lab and lives as my doctoral education migrated across st ate boundaries. The experience I have acquired at Michigan State University through both of yo u has been an integral part of my personal and professional development. Hasand Gandhi, Erich Bartels, and Jeff Landgraf also deserve more accolades than I can possibly provide. This research would have never come to fruition witho ut their support and guidance in the laboratory. I also thank Dr. James Cervino for his unwavering support of this project. My friends, which now span the globe, also deserve recogni tion. I hope that our shared experiences have benefited you all as much as they have enriched my l ife. Briana Hauff , I thank you most of all, not only for accepting the chall enge of this project with me but also for your support in overcoming the countle ss hurdles we experienced together . v TABLE OF CONTENTS v ii vi ii CHAPTER 1 DECOUPLED SEASONAL S TRESS AS AN INDICATI ON OF CHRONIC STRESS IN MONTASTRAEA CAVERNOS A AND PORITES ASTREOIDES INHABITING THE FLORI DA REEF TRACT 1 1.1 Abstract 1 1.2 Introduction 2 1.3 Materials and M 5 1.3.1 Coral communities of the patch reef and bank reef s ystems 5 1.3.2 Coral community characteristics at inshore and offshore reefs 7 1.3.3 Location - dependent colony brightness, a bleaching characteristic 8 1.3.4 Statistical analyses 9 11 1.4.1 16 - year comparison of zonal coral communities 11 1.4.2 Zonal abiotic characteristics 20 1.4.3 Location - dependent shifts in brightness, a bleaching related characteristic 25 30 3 5 36 LOCAL ADAPTATION OF PORI TES ASTREOIDES BETWEEN INSHORE AND OFFSHORE METAPOPULAT IONS INHABITING THE FLORI DA REEF TRACT 36 36 38 41 43 44 2.3.3 Two - step qRT - 45 2.3.4 qRT - 45 2.3.5 qRT - 47 49 2.3.6.1 Tumo r necrosis factor receptor asso 49 2.3.6.2 Adenylate cyclase associated protein 2 (ACAP2) 49 2.3.6.3 49 2.4 50 2.4.1 Site ..... ................. 50 2.4.2 Effects of site and season on pooled GOI transcript abundance 52 2.4.3 Gene specific res 54 54 2.4.3.2 e 58 59 2.4.4 Summa ry of factors affecting host gene expression in P. astreoides 60 vi 62 2.5.1 Activation of host coral immune pathways: TN F receptor associated factor 3 expression 62 2.5.2 Cellular stress response: e ukaryotic translation initiation factor 3, subunit H (eIF3H) 65 2.5.3 Adaptive response to immune system activation: a denylate cyclase associated protein 2 (ACAP2) 67 69 71 DIVERGENT RESPONSES OF PORITES ASTREOIDES POPULATIONS TO BACTE RIAL ENDOTOXIN : P OTENTIAL CONSEQUENCES OF IMMU NE SYSTEM ACTIVATION 71 71 73 76 76 79 80 81 3.3.5 Two - step qRT - 81 3.3.6 qRT - 82 3.3.7 qRT - PCR 84 85 85 8 8 3.4.3 Synergistic effect of temperature and lipopolysaccharide (32°C + 5µL mL - 1 LPS) 89 90 100 WORKS CITED 101 vii LIST OF TABLES Table 1: Selection C r iteria for Primers Used to Amplify Transcripts for Quantitative Real - Time Polymerase Chain R eaction 46 Table 2: The Amplification Efficiency, Primer Sequences of Each Gene of Interest, and Control G enes a re Presented in the T able 47 Table 3: Seawater Temperatures Reported at the Date of Collection at the Offshore S ite (Acer24 Reef) and the Inshore Site (Birth day Reef) 51 Table 4: Two - Way Factorial Mixed Effects Model for Transplant Site - Dependent and Seasonal Effects on Transcript A bundance of Porites astreoides Following Reci procal Transplantation Between an Inshore Patch Reef (Birthday Reef) and O ffshor e Bank R eef (Acer 24 Reef ) 55 Table 5: Two - Way Factorial Mixed Effects Model for Collection Site - Dependent and Seasonal Effects on Transcript A bundance of Porites astreoides Following Reciprocal Transplantation Between an Inshore P atch Reef (Birthday Reef) and Offshore Bank R eef (Acer 24 Reef ) 57 Table 6: The Amplification Efficiency and Primer Sequences of Each Gene of Interest an d Control Gene viii LIST OF FIGURES Figure 1: Map of Lower and Middle Florida Keys Study Sites 6 Figure 2: Annual Percentage (%) of Scleractinian Coral Cover from 1996 - 2011 12 Figure 3: Mean Rényi Diversity Profiles of CREMP and Established Patch and Offshore Reef Sites 13 Figure 4 : Principal Coordinate Analysis of Species Occurrence from 1996 - 2011 14 Figure 5: The Mean Percent Cover of Benthic Substrata at Birthday and Acer24 Reefs .. 16 Figure 6: The Mean Abundance and Area of Corals in Transects of Established Sites 18 Figure 7: The Frequency Distributions of Si zes for all Corals and Three Selected Species at Established Sites 19 Figure 8: Principal Component Bi - plots of Winter Environmental Data from Patch Reef and Offshore Reef Sites from 1996 - 2 011 22 Figure 9: Principal Component Bi - plots of Summer Environmental Data from Patch Reef and Offshore Reef Sites from 1996 - 2011 23 Figure 10: Frequency of Occu rrence for Three Brightness States Following a Reciprocal Transplant of Porites astreoides and Montastraea cavernosa 26 Figure 11: Quantum Yield of Photochemical Energy Conversion of Montastraea cavernosa and Porites astreoides Inhabiting an Offshore Reef (Acer24) or an Inshore Reef (Birthday) within the Florida Reef 27 Figure 12: Brightness Time Series of Reciprocally Transplanted Montastraea cavernosa and Porites astreoides from September 2011 - April 2013 29 Figure 13: The Lower Region of the Florida Keys is Presented Along with Several Orientating L ocations 42 Figure 14 : The Figure Displays Hourly Water Temperature for Birthday Reef (Inshore Patch Reef) and Acer 24 (Offshore B ank Reef) Over the Course of the Two - Year Reciprocal Transplantation E xperiment 5 1 Figure 15: Principal Coordinate Analysis Results Indicating a Collection Site - Dependent Effect on Transcript A bundance 53 ix Figure 16: The log2 Scaled Abundances of TRAF3 Transcripts from Corals Reciprocally Transplanted Between an Inshore Site (Birthday Reef) and an Offshore Site (Acer 24 Reef ) . 55 Figure 17: The log2 Scaled Abundances of e IF3H Transcripts from Corals Reciprocally Transplanted Between an Inshore S ite ( Birthday Reef) and an Offshore Site (Acer 24 59 Figure 18: The log2 Scaled Abundances of ACAP2 Transcripts from Corals Reciprocally Transplanted Between an Inshore Site (Birthday Reef) and an Offshore S ite (Acer 24 60 Figure 19: The Inshore Patch R eef (Birthday Reef; - ) and Offshore Bank R eef (Acer 24 Reef; - ) Sampling Sites are Pictured A bove 77 Figure 20: e IF3H Transcript Abundances A ssociated W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef Site Following 8 h Incubation With One of Three Treatments; 28°C (Control) (28) , 32°C (32) , and 32°C + Lipopolysaccharide (32L) of Serratia marsescens 85 Figure 21: HSFP1 Transcript Abu ndances Associated W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef Site Following 8 h I ncubation With One of Three Treatments; 28°C (C ontrol) (28) , 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens 86 Figure 22: TRAF3 Transcript Abundances Associated W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef S ite Following 8 h Incubation W ith One of Three Treatments; 28°C (C ontrol) (28), 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens 87 Figure 23 : CDPK Transcript Abundances Associated W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef Site Followi ng 8 h Incubation With One of Three Treatments; 28°C (C ontrol) (28), 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens 88 Figure 24 : The Mean Gene E xpression Response Strategies to Temperature and L ipopolysaccharide of Porites astreoides Inhabiting Inshore Patch Reefs an d Offshore Bank R eefs of the Florida Reef Tract 97 1 CHAPTER 1 DECOUPLED SEASONAL S TRESS AS AN INDICATI ON OF CHRONIC STRESS IN MONTASTRAEA CAVERNOSA AND PORITES ASTREOIDES INHABITING THE FLORI DA REEF TRACT 1.1 Abstract Scleractinian coral abundance and diversity increases along inshore to offshor e transects across the Florida Reef T ract (FRT ) . I identify this trend among coral reefs throughout the middle and lower Florida Keys Region from Coral Reef Environmental Monitoring Program (CREMP) datasets as well as an inshore and offshore reef at similar depths. Although mass mortality from disease and climate anomalies are largely to blame for rapid losses in offshore reef cor al cover, the failure of extant coral populations to recolonize this zone is puzzling given improvements to water quality and the mild seawater temperature regime compared to corals inhabiting the patch reef zone. Applying ex ploratory statistical methods I identified two abundant species, Montastraea cavernosa and Porites astreoides , inhabiting both zones to varying degrees. Following reciprocal transplantation of conspecifics between a representative bank reef and patch reef zone (6 m depth), I monitored monthly coral colony brightness (a measurement related to algal symbiont density) over a two - year period to examine symbiont loss, a common stress response in scleractinian corals. Although species - specific stress patterns were not identified, zone - speci fic variation was evident. Trigonometric regression of stress level by month revealed a significant relationship supporting an annual stress and recovery period at the inshore patch reef zone. Contrary to this result, conspecifics transplanted to the off shore zone followed a positive linear trend indicating the absence or diminishment of a recovery phase resulting in continued chronic bleachi ng over the two - year period. My results implicate the importance of turbidity in alleviating stress at inshore site s and the importance of extending greater protection to reefs within this zone. 2 1.2 Introduction The Florida Keys Reef Tract has experienced dramatic decreases in coral cover (Aronson and Precht, 20 01) , shifts in reef communities (Pandolfi et al., 2005; Ruzicka et al., 2013) , and decreases in reef architectural complexity (Alvarez - Filip et al., 2009) , which have all resulted in decreased biodiversity. This trend is most apparent along the 15 offshore reef preservation areas of the Florida Keys National Marine Sanctuary. Located between 1 and 8 m depths and 5 to7 km offshore, these once highly productive reef crest and fore reef ecosystems no longer support accreting scleractinian coral communities and can be considered relict in comparison to historical baselines (Palandro et al., 2008) . This imperiled status of the offshore reef zone is not a result of a single stressor. Diseases and macro - alg al overgrowth arising from human interaction, decreased coral resilience following destructive hurricanes and anomalous hypothermic (Hudson et al., 1976; Shinn et al., 2003; Walker et al., 1982) and hyperthermic seawater temperature events have all greatly affected this region. Presently, conditions are such at offshore reefs that corals should display net accretion, however reef building coral populations remain at low abundances. Observing transects extending from inshore to offs hore, coral abundance decreases to the offshore bank reef zone (Lirman and Fong, 20 07; Roberts et al., 1982; Schutte et al., 2010) with approximately two - times lower abundance at offshore reefs. In contrast, sustained growth has been observed throughout the inshore patch reef system despite far fewer protected areas in contrast to of fshore reefs, increased frequency of hyper and hypothermic seawater temperature events experienced (So to et al., 2011) , increased dissolved inorganic nitrogen, soluble reactive phosphorus, and chlorophyll a, increased turbidity, greater macro algal biomass (Lapointe et al., 2004) , and closer proximity to anthropogenic sources of stress (Ginsburg et al., 2001) . Several hypotheses have been proposed regarding the proliferation of inshore reefs and the deterioration of offshore reefs including (1) conti nued persistence of large fecund colonies in near - shore reef zones resulting in increased near - shore recruitment, (2) diet supplementation from increased turbidity and 3 nutrient concentration as a means to alleviate stressful near - shore conditions, and (3) differences in seawater temperature variance resulting in a wider temperature range allowing inshore reefs an opportunity to experience a broader range of temperatures acclimation (Soto et al., 2011) . Recruitment does not appear to be responsible for the differences in coral abundance between adjacent inshore and offshore sites. The current state of knowledge emphasizes the importance of local recruitment for the maintenance of coral population abundance (Mumby and Stenick , 2008; Steneck et al., 2009) and low frequency immigrant settlement promotes genetic diversity (Noreen et al., 2009; Sammarco and Andrews, 1989) . Co - implementation of larval dispersal models, genetic population connectivity (Foster et al., 2012) and sensitive identification and quantification of recruits (Hsu et al., 2014; Schmidt - Roach et al., 2008) has increased the accuracy of coral recruitment estimation. In the Florida Keys annual recruitment at inshore and offshore reefs is hig hly variable (Moulding, 2005; Shearer and Coffroth, 2006) and not a locally defining characteristic (Miller et al., 2000) . Post settlement processes and stress (i.e. hypotheses 2 and 3) are therefore more likely correlated to the difference in reef growth and community structure observed. I hypothesize that corals inhab iting inshore reef zones are able to respond to a wider range of stress levels as a consequence of frequent occurrences of non - lethal stress characteristic of this zone. The history of abiotic and biotic stress experienced by a reef affects the inhabitant s response, growth, selection for resistant individuals or in the case of corals establishment of resistant symbionts (Haslun et al., 2011; Oliver and Palumbi, 2011) following aberrant levels of stress. Gradients of increased levels of abiotic factors associated with stress extend from offshore reefs to inshore reefs in the Florida Keys (Lapointe et al., 2004) and supporting this hypothesis a negative correlation exists when between both coral cover and colony size, and distance from shore (Lirman and Fong, 2007) . Although gradients may 4 exist along this transect the detrimen t of each potential stressor (e.g. nutrients, temperature, and light) to a species may not be biologically relevant despite significant location - dependent differences. Further , the stress level experienced by an organism is most likely not a reflection of any one stressor but rather the cumulative effect of stressors present. The level of one factor may provide a refuge from a factor the organism is more susceptible to decreasing the severity of a potentially lethal stressor. For example, increases in ch lorophyll a occur concomitantly with increases in turbidity. Increased turbidity may in - turn, decrease photic stress on scleractinian corals during increased water temperatures resulting in an overall decreased level of stress. Identification of abiotic f actors contributing to the community level differences in abundance and size of corals inhabiting inshore and offshore reefs is critical to the management of this habitat (Lirman and Fong, 2007) . This study integrated and applied coral cover data from the coral reef environmental monitoring project (CREMP), environmental data from the water quality monitoring project (WQMP), and coral community and environmental data from ongoing research projects to describe the asymmetry of coral cover currently observed between the inshore and offshore bank reefs . I identify four coral species with differential abundances within the nearshore and offshore zones, the study of which may increase our un derstanding of the differences in coral growth between these environments. To provide a more complete understanding of the community dyn amics of a symbiotic organism I also observed the seasonal photosynthetic competence (symbiont photo - physiology) and colony color of the identified species at two sites, one representative of an inshore patch reef and the other representative of an offshore bank reef, over a two - year period. From this information the seasonal response strategy of each s pecies was determined. 5 1.3 M aterials and Methods 1.3.1 Coral communities of the patch reef and bank reef s ystems Relative abundances of corals inhabiting offshore shallow and middle channel patch reefs, spanning a 16 - year period, were obtained from the Coral Reef Evaluation and Monitoring Project (CREMP). This dataset has been applied to the detection of potential causal factor (s) for regional differences in benthic communities coupled with Florida Keys National Marine Sanctuary (FKNMS) water quality mo nitoring project (WQMP) (Maliao et al., 2008) . Maliao et al. (2008) also determined that the CREMP monitoring strategy correlates well with the results of other sampling efforts including the Atlantic and Gulf Rapid Assessment protocol (AGRRA). I analyzed CREMP and WQMP datasets spanning a 15 - year period from 1997 - 2012 to determine the variability in the communities of corals and environmental regimes associated reefs grouped as either offsho re bank reef or as part of the inshore patch reef rather than at the regional scale. Of the available reefs within the two datase ts I selected reefs within the lower and middle keys for three reasons. Firstly, throughout the lower and middle keys regions there are a number of offshore bank reefs as well as patch reef formations that spatially parallel one another (Figure 1). Secon dly, for the past three years I monitored an inshore patch reef and an of fshore bank reef centralized within this range , Birthd ay and Acer 24 reefs respectively (Figure 1). Thirdly, this region has been characterized by decreasing gradients of nutrients, turbidity and temperature from the inshore to offshore zones. Differences in these factors among sites and between the inshore and offshore zones (i.e. nutrients, turbidity, and temperature) may provide a foundation to understand the evident variation in coral cover. 6 Figure 1: Map of Lower and Middle Florida Keys Study Sites. Sites were either selected from the Coral Reef Enviro nmental Monitoring Project (Diagonal Labels) or from sites established for this study (Horizontal Labels). Sites identified with open triangles indicate offshore sites (n = 6) while those wit h open circles indicate inshore patch reef sites (n = 6) . Reefs with greater than 10% total coral cover as of 1996 were included. Reefs with cover less than 10% as of 1996 may have already become relict or may never have supported a large community of corals and were therefore excluded. From this criteria, five C REMP sites remained as representative of each of the two zones. Inshore sites included Western Head, Cliff Green, West Washerwoman, Dustan Rocks (also referred to as East Washerwoman), and Western Turtle Shoal. West Washerwoman is the largest patch reef o f this set and is considered the largest in the lower keys. East Washerwoman and Western Turtle Shoal are also considerably large patch reef groups while Western Head and Cliff Green are singular patch reefs (see Lidz et al. (2007) for mapping and descript ion of these patch reefs). Offshore shallow reefs included Sand Key, Rock Key, Eastern Sambo, Looe Key, and Sombrero Key. Each of these offshore bank reefs is characterized by spur and groove formations preceded inshore by patch reefs. 7 1.3.2 Coral commu nity characteristics at inshore and offshore r eefs Colony size and abundance of corals were assessed biannually from 2011 to 2013 using photo - transects - - Assessments were conducted by divers using self - contained underwater breathing apparatus (SCUBA) during winter (February) and summer (August) seasons. Divers ph otographed the benthos at random non - overlapping points (n = 15) along 30 m transects (n = 3) with a Nikon D5100 camera affixed to a 0.5 x 0.5 m quadrapod (Coyer et al., 1999). Lighting was supplied by a Fantasea NanoFlash strobe. Transects were measured using surveyor tape and extended out from a central location in three randomly assigned directions for each season. The photo - quadrat method has been shown to be the most cost effective method to assess coral and sessile benthic communities without sacrif icing accuracy (Leujak and Ormond, 2007) . Images were analyzed using Coral Point Count with Excel (CPCe) extensions (Kohler and Gill, 2006) . Coral species richnes s, abundance, area, percent cover, as well as benthos composition including coral, sponge, algae, sand, and rubble were determined. Further, all individual Montastraea cavernosa and Porites astreoides colonies within 1 m of transect were analyzed for phot osynthetic capacity using a Diving Pulse Amplitude Flourometer (PAM: Walz). I was unable to assess field corals in a dark adapted state and therefore obtained the effective quantum yield of photochemical energy conversion ( m . The change in F ( F) r epresents the change in fluorescence from the maximal fluorescent yield of the coral in an illuminated environment (F m ) following a saturating pulse from the instrument. The use of these two measurements has been found to be highly correlate with quantum yield (Genty et al., 1989) . The effective quantum yield was determined at the apex of colonies with the fiber quantum se nsor held within the Surface Holder, to maintain a distance of 10 mm between it and the colony tissue (Cervino et al., 2012) . 8 1.3.3 Location - dependent colony brightness, a bleaching c haracteristic Following the identification of species characteristic of the inshore patch reef and offshore bank reef systems, a reciprocal transplant was carried out between Birthday Reef and Acer 24 with two species of coral, M. cavernosa and P. astreoides . Coral frag ments (16 x 16 cm) of both species were collected from colonies at least 10 m apart at each reef using a steel mallet and cold chisel. Permission for field work at Birthday Reef and Acer 24 was granted by National Oceanic and Atmospheric Administration Na tional Marine Sanctuaries (Permit # FKNMS - 2011 - 107). Fragments were transferred in large coolers filled with site - derived water to the Mote Marine Laboratory Tropical Research Laboratory (MML), where they were immediately sectioned into two 8 x 8 cm fragme nts with a tile saw lubricated and cooled with sterile artificial sea water (Instant Ocean) sprayed on the blade with a wash bottle. Following sectioning, fragments were transferred to MML flow - through seawater raceways. Raceways were shaded from direct su nlight to decrease stress. Following 2 days of recovery, fragments were attached to pucks (1 part concrete: 3 parts aragonite sand) with a two - part epoxy (All Fix Epoxy; Philadelphia, PA USA). After another 3 days of recovery, corals were transferred in la rge coolers filled with seawater from the MML flow - through system, to each field site. Each field site consisted of 6 concrete blocks affixed to the calcium carbonate substrate using a 1 part plaster: 2 parts concrete mixture in a hexagonal shape. Each con crete block consisted of 6 - 7 randomly assigned coral fragments of a single species. Fragments were attached to each block with All Fix Epoxy. Neighboring blocks harbored different species. Images of each cinder block and its associated coral fragments, were taken monthly over the course of two years with a Nikon D500 in a Fantasea underwater housing. Illumination was provided by the built - in camera flash and housing diffuser. Each image was analyzed with Image J to determine the mean fragment brightne ss. The brightness of gray - scale images is highly correlated with symbiont density (Siebeck et al., 2006) and thus, colony darkening (decreased brightness) reflects increased 9 concentrations of the coral end osymbiont (Siebeck et al., 2006) . Analysis proceeded by first transforming an image to 8 - bit format. The scale of each image was set using the average width of a cinder block (15.24 cm). Regions were drawn around each fragment including only live c oral tissue using the polygon tool and minimum, maximum, and mean brightness values obtained. Differences in the background brightness between different blocks and/or site dependent water quality (turbidity) was corrected for by adding the corresponding v alue or subtracting the corresponding value to reach that of the first sampling period for each block. 1.3.4 Statistical a nalyses Statistical analyses were performed with R v ersion 3.0.2 (Team, 2013) and all figures were cre ated with ggplot2 (Wickham, 2009) . We first analyzed the CREMP dataset for variation in coral cover among sites and zones across the 16 - year sampling period via two - way repeated measures analysis of variance (ANOVA). The aov() function within the statistical p ackage R was applied with reef zone and sites as predictors of total coral cover and an error term described by sample year to create a repeated measures design. Assumptions of ANOVA were visually evaluated with the plot() function, which produces figures including residuals vs . the fitted model, standardized residuals vs . theoretical quantiles (qq - plot), square root of the standardized residuals vs . the fitted values, and the standardized residuals at each factor level. Non - linearity in these figures is in dicative of a deviation from a normal distribution. Homogeneity of variances was assessed using the Bartlett test (bartlett.test()), which tests the null hypothesis that variances are equal at all levels. 10 Site community richness and evenness was assessed across the 16 - year CREMP dataset and the 2 - year dataset from our field sites using Rényi diversity profiles (Jost, 2006) . The renyi () function from the vegan package served as the platform for this analysis (Oksanen, 2013) . Mean relative abundances of coral species across the 15 - year pe riod were applied to this function. The shape of each profile provides information with respect to the evenness of a community. Horizontal lines represent perfectly even communities where all species are present in equal abundances. Alpha values of 0, 1, 2 , and infinity indicate species richness, Shannon diversity index, Simpson index (reciprocal), and the proportion of the most abundant species present, respectively. Coral community differences were identified using a multivariate approach. Principal coor dinate analysis (PCoA) was performed on the annual relative abundances of coral species at each site. To accomplish this, Bray - curtis dissimilarities were calculated with the vegdist () function from the vega n package (Oksanen, 2013) . Eigen analysis was performed by the application of the cmdscale () function on this dissimilarity matrix in 2 dimensions to produ ce eigen values. Confidence intervals (95%) were calculated from these eigen values across years for each site. The correlations between the original relative abundances and eigen values were used as loadings to determine the association of species with p articular sites. Ad - hoc contrasts were used to identify differences between transplant groups, within a general linear model framework. Contrasts included (1) month dependent shifts in brightness between corals transplanted to and from the site of origin and (2) site and t ransplant dependent shifts in the mean brightness of a species. Seasonal patterns in mean brightness were identified with trigonometric linear modeling, applying a period of 12 to accommodate our monthly data interval. 11 1.4 Results 1.4.1 16 - year compariso n of zonal coral c ommunities Analysis of the CREMP dataset revealed that coral cover declined at 8 of 10 reefs between 1996 and 2011 (Figure 2). Decreases in coral cover were greatest between 1999 and 2000. Coral cover declined from 25 - 15 % at the three western - most middle channel sites (Western Head, Cliff Green, and West Washer Women) between 1999 and 2000 followed by marginal loss or stability in the remaining coral cover. The eastern most sites, Dustan Rocks and Western Turtle Shoal, maintained coral cover during this period while coral cover at all offshore bank reef sites decreased significantly between 1996 and 2000. Despite similar trends of coral cover loss, the total mean coral cover was significantly greater at the monitored inshore patch reef s compared to offshore bank reefs (RM - ANOVA: p < 0.0001). In addition to the significant differences in coral cover, we identified community dissimilarities between offshore shallow and middle channel patch reefs. Species richness was significantly greater at middle channel sites (ANOVA: p < 0.05), with 9 additional species present when rare species were considered. Removing species accounting for less than 0.1 % of the mean benthos cover resulted in a single species difference between the zones. R é nyi profiles of coral abundance indicated that all sites were dominated by a few species and therefore, are characterized by low species evenness (Figure 3). The most speciose sites, Looe Key from offshore reefs and Western Head from inshore reefs, displ ayed the least evenness. Offshore sites other than Looe Key reef, displayed similar profiles, indicated by the similar slopes of R é nyi profiles. Although middle channel sites displayed decreased variation in species richness, diversity profiles indicate a range of characteristics. Dustan Rocks and Western Washer Woman were the most similar of the middle channel sites while Cliff Green with very similar richness to these two sites, was less even. 12 Figure 2: Annual P ercentage (%) of Scleractinian Coral C o ver from 1996 - 2011 . Offshore shallow reef sites are grouped along the top row while inshore patch reef site s are grouped along the bottom row . WHead = Western Head, CGreen = Cliff Green, WWWomen = Western Washer Women, DRocks = Dustan Rocks, WTShoal = West Turtle Shoal, ESShallow = Eastern Sambo Shallow, LKShallow = Looe Key Shallow, SShallow = Sombrero Shallow, RKShallow = Rock Key Shallow, SKShallow = Sand Key Shallow. See figure 1 for site location. 13 Figure 3: Mean Ré nyi Diversity P rofiles of CREMP and Established Patch and Offshore R eef S ites. Patch reef points (grey triangles) and offshore points (black circles) are identified similarly for CREMP data (A) and established sites (B). CREMP data points reflect the mean value at each location across the s ampling period from 1996 - 2011, while points for established sites reflect mean Rényi values of transects carried out in the summer and winter of 2012 (n = 6 per site). Loess regression was applied to visualize each trend. Zonal ordination of coral communities was identified following PCoA (Figure 4). Middle channel sites ordinated closely together in quadrant 3 while offshore shallow reefs, although displaying more variability, ordinated in the periphery away from this clu ster in quadrants 1, 2, and 4, centering around quadrant 2 (Figure 4). Evaluating the loadings, along the first two axes, the abundances of some coral species were dissimilar between locations. Inshore patch reefs were characterized by an increased preva lence of Montastraea cavernosa and Sidereastrea siderea , while Porites astreoides and Acropora palamata were more common at offshore bank reefs. 14 Figure 4: Principal Coordinate Analysis of Species O ccurrence from 1996 - 2011. The scores for each site (A) and loadings for each species (B) following principal coordinate analysis are presented. The first two coordinates accounting for 64.5 % of variation in the data are plotted. Error bars (A) indicate 95 % confidence intervals across the 15 - year period. Spec ies present in a particular quadrant of the loadings plot are more likely to be associated with a site in the same quadrant of the score plot. Analysis of CREMP data supports the ongoing observation of increased growth at inshore patch reefs, however this data set is limited to relative abundance. To circumvent this issue we selected two reference sites to analyze in greater detail, focusing on coral community characteristics, particularly colony abundance, colony size, and benthic community composition. The mean seasonal benthos composition at Acer 24 reef (an offshore reef) and Birthday reef (a patch reef) along 30 m transects is presented in Figure 5. Scleractinian coral cover was significantly greater at Birthday Reef (ANOVA: p < 3.83e - 07), while the benthic community at Acer 24 contained significantly greater gorgonian coral cover (ANOVA: p < 2.01e - 08), supporting our observations from the CREMP data analyses. Gorgonian cover 15 also displayed seasonality at Acer 24 reef, with a greater percent cover observed during winter sampling (ANOVA: p < 0.003). This pattern was not evident at Birthday reef. Macro algal and turf algal cover was not sig nificantly different between the reference sites and accounted for greater than 30 % of all reef cover. Increased macro algal cover was observed during winter months, but this difference was not significant compared to summer. 16 Figure 5: The Mean Percent Cover of B enthic S ubstrata at Birt h day and Acer24 R eefs . Benthic substrate information was collected during the summer (August) and winter (February) from 2011 - 2012 along 30 m tran sects (n = 6). Error bars represent 95 % confidence intervals. 17 dissimilarity between the two sites. Rényi diversity profiles support th e dissimilarity in scleractinian coral community composition (Figure 3). Species richness was greater at Birthday reef as indicated by an increased diversity value at alpha = 0. Fourteen scleractinian coral species were present at Birthday reef while onl y ten of these species were observed at Acer 24 reef. Additionally, both sites were characterized by low species evenness indicated by diversity values > 1 for alpha = Infinity. The most frequently observed colonies inhabiting Acer 24 reef were Siderastr ea siderea , Montastrea cavernosa , Porites astreoides , and Stephananocoenia michelinii (Figure 6). In addition to these four species, Orbicella annularis was frequently observed at Birthday reef. Larger colonies were more frequently observed at Birthday R eef, as indicated by the greater positive skew of total coral frequency distributions (Figure 7). Amongst the most common species identified, P. astreoides , S. siderea , and M. cavernosa all occurred in similarly comparable zone dependent distributions. 18 Figure 6 : The Mean A bundance and A rea of Corals in Transects of Established S ites. Bars indicate the mean count of a particular species of coral. Error bars represent the standard error for each bar. The color of each bar represents the mean area (cm - 2 ) along transects for each species at a given site. Acer 24 is representative of an offshore site while Birthday reef is representative of a patch reef. 19 Figure 7 : The Frequency Distributions of Sizes for all Corals and Three Selected Species at Establis hed S ites. The four figures show the total coral colony size distribution (A), as well as the size distributions of Porites astreoides (B), Montastraea cavernosa (C), and Siderastrea siderea (D), the three most abundant species. 20 1.4.2 Zonal abiotic c hara cteristics Principal component analysis (PCA) was applied to winter and summer subsets of the WQMP dataset to identify factors that may be associated with stress during these periods and influence the coral communities of inshore and offshore reefs. Durin g winter months (January and February) 55 % of the variation between sites was accounted for by three components. Variation along principal component 1 (22.9 %) was influenced by water chemistry attributes. Total organic nitrogen (TON) ordinated sites ne gatively while the inorganic nutrients ammonium and silicate ordinated sites positively along this axis. These three variables did not vary significantly by region or location, however the marginal assurance in variation associated with silicate (ANOVA; p = 0.0652) provides a potential difference between patch and offshore reefs during winter months. Along principal component 2 (19.2 %), variation between sites was dictated by nitrate, soluble reactive phosphorus (SRP), chlorophyll a (Chl a), and total or ganic carbon (TOC). Nitrate was significantly greater at eastern sites than western sites and independent of location (ANOVA; p = 0.0062), while SRP, Chl a, and TOC remained statistically similar across regions and locations. The variation along principa l component 3 (13.1 %) was dictated by the variation in the physical characteristics turbidity and temperature. Turbidity was significantly greater at western sites regardless of location (ANOVA; p = 0.0113), while seawater temperature (SWT) did not was n ot significantly different for either region or location. Additionally, during winter months, greater variation in SWT was observed at inshore reefs (18 - 29 ° C) than on offshore reefs (20 - 25 ° C). Plotting each of these components against one another resulte d in regional ordination (Figure 8). With the exception of Rock Key, the western most sites West Washer Woman, Eastern Sambo, Cliff Green, Western Head and Sand Key, oriented away from eastern sites: Looe Key, Sombrero Reef, Dustan Rocks and Western Turtl e Shoal. Variation in SWT, turbidity, and TOC appear responsible for the observed region dependent ordination during winter months, indicated by the direction and magnitude of arrows 21 for each environmental variable. Within this structure, the three most speciose inshore sites, Western Head, Cliff Green, and West Washer Woman ordinated near one another. Principal component analysis of summer WQMP data indicated 48.8% of the environmental variability among all sites could be explained by the first three components. Similar to PCA of winter months, regional ordination of sites was evident during the summer (Figure 9). The three speciose western sites Western Washerwoman, Cliff Green, and Western Head ordinated away from the eastern sites Sand Key, Rock K ey and Sombrero Reef, and Looe Key, while the remaining sites, Eastern Sambo, Western Turtle Shoal, and Dustan Rocks were found between these two groups. This separation was not a distinct as that identified during winter months and may provide an indicat ion of the large potential for site or location dependent environmental variability during summer months for reefs of the middle and lower Florida Keys. 22 Figure 8 : Principal Component Bi - plots of Winter Environmental Data from Patch Reef and Offshore Reef S ites from 1996 - 2011 . Three bi - plots represent combinations of the first three components representing 55 % of the variation in the dataspace; component 1 - compone nt 2 (A), component 1 - component 3 (B), and component 2 - component 3 (C). Site names are positioned at the score associated with that site. Red arrows indicate the loading of a given environmental variable on the ordination of sites. The length and directio n of the arrow is proportional to its effect. 23 Fig ure 9 : Principal Component B i - plots of S ummer Environmental Data from Patch Reef and Offshore Reef S ites from 1996 - 2011 . Three bi - plots represent combinations of the first three components representing 4 8.8 % of the variation in the dataspace; component 1 - component 2 (A), component 1 - component 3 (B), and component 2 - component 3 (C). Site names are positioned at the score associated with that site. Red arrows indicate the loading of a given environmental variable on the ordination of sites. The length and direction of the arrow is proportional to its effect. 24 Despite explaining only 13.3 % of the total variation, PC3 appeared largely responsible for the regional ordination of sites within the data - space. Clear ordination was evident only in bi - plots including this component as an axis. Therefore the environmental variables TON, ammonium, and SWT, which were most influential along PC3, may be important factors governing summer environmental differences bet ween these regions and sites. Upon closer inspection, SWTs did not vary significantly between regions, however inshore sites were significantly warmer than offshore sites by 1 ° C (ANOVA; p = 0.0183). Further, SWT at inshore sites was consistently warmer d uring summer months (32 - 27 ° C), while offshore site SWTs were typically cooler but also ranged more widely than inshore sites during this time (31 - 20 ° C). Significant differences were not identified at the location or region level for TON (ANOVA; p = 0.166 and p = 0.214 respectively) or ammonium (ANOVA; p = 0.42 and p = 0.06 respectively). Principal components 1 and 2, despite limited influence on the regional ordination of sites, imparted greater percentages of variation to the summer environment data - sp ace than PC3. Influential variables along principal component 1 (20.2 %) included factors that contribute to turbidity including Chl a and silicates. Western sites were 0.7 NTU more turbid than eastern sites (ANOVA; p = 0.022) and also significantly grea ter in Chl a (ANOVA; p = 0.013). Silicates displayed significant variation at the zone level (ANOVA; p = 0.0392) but not regionally. Variation along PC2 was influenced by SRP, nitrate, and TOC, however significant variation was not identified for any of these variables at the regional or location level. Increasing the resolution of temperature data to daily monitoring at a reference inshore and offshore site provided greater support to these findings. During three years of temperature monitoring (2011 - 2013) annual mean seawater temperatures (SWT) at the inshore site, Birthday Reef, and offshore site, Acer 24 reef, did not significantly differ (ANOVA p = 0.828); 26.74°C and 26.77°C respectively. Monthly 25 variance in SWT, however was 0.5°C less at Acer 24 (ANOVA p < 0.05). Additionally, the frequency of da ily SWTs greater than 30°C and less than 23 ° C were 44 % and 50 % more frequent at Birthday reef than Acer 24. 1.4.3 Location - dependent s hifts in brightness, a bleaching related c haracteristic Fragments of M. cavernosa and P. astreoides were significantly darker in brightness at Birthday Reef (ANOVA; p = 2e - 16 ), the inshore site, relative to the offshore site Acer 24 (Figure 10). Although the occurrence of severely bleached corals (Brightness 150) was rare, the frequency of stressed c orals (101 Brightness 149) was significantly greater at the offshore site, Acer 24. We did not observe a transplant dependent effect on the brightness of corals when analyzing the total frequency of occurrences at the three brightness ranges. Observi ng the quantum yield of photochemical energy conversion (Yield: F/F m species (p = 2.23e - 06 ) and site (p = 0.0198) levels during summer months (Figure 11). The probability of ph otons entering photosystem II (PSII) was greater for P. astreoides and colonies of these two species inhabiting Birthday reef. During the winter, yields did not vary significantly from that of summer months indicating similar levels of photic stress. 26 Figure 1 0: Frequency of Occurrence for Three Brightness States Following a R eci procal T ransplant of Porites astreoides and Montastraea cavernosa . Three bins were created for mean brightness values, each spanning approximately 40 units. T he four graphs are separated along the y - axis by the site to which a coral was transplanted. Bar color indicates collection site (Black = Acer 24, Grey = Birthday). The graphs are further separated by species along the x - axis. Bar height is indicative of t he cumulative number of occurrences within a particular bin from monthly images taken from September 2011 - April 2013. 27 Figure 11: Quantum Y ield of Photochemical Energy C onversion of Montastraea cavernosa and Porites astreoides Inhabiting an Offshore Reef (Acer 24) or an Inshore R eef (B irthday) within the Florida Reef Tract. The yields of all corals of the two species within 1 m of 90 m transects (n = 3) were obtained for a given sampling event. Points represent means and erro r bars indicate 95 % confidence intervals. I observed a seasonal change in the monthly brightness of coral fragments transplanted to Birthday Reef, the inshore site, following the application of a Loess smoothing function (Cleveland and Devlin, 1988) . The lightest shade (greater brightness value) was observed during September and October while the darkest (lower brightness value) were observed during February and March. Due to the large variance in monthly brightness values between coral fragments, trigono metric linear regression was applied to determine if a significant trend existed. Changes in brightness significantly fit a cosine function with 12 - month period (ANOVA; p = 0.04), representative of a significant annual pattern in coral brightness at Birth day reef (Figure 12). A significant cosine pattern was not observed for corals at Acer 24. Instead, linear regression of coral fragment brightness at this site indicated that mean brightness 28 values of corals transplanted to this site increased from Septe mber 2011 to May 2013. Therefore these corals became progressively lighter (more bleached) during this period of time. 29 Figure 12: Brightness Time Series of Reciprocally T ransplanted Montastraea cavernosa and Porites astreoides from September 2011 - April 2013. The monthly brightness value of each individual coral within the transplant study is presented. Points identify the site a co ral was transplanted to (Circle = Acer 24, Triangle = Birthday). Loess regressio n lines have been fitted to each subset of corals also based upon the site a coral was transplanted to (Line = Acer 24, Dash = Birthday). Confidence intervals (95 %) have been place around each line. The four graphs are separated along the y - axis by collec tion site and along the x - axis by species. 30 1.5 Discussion The inshore patch reef system of the Florida Keys National Marine Sanctuary (FKNMS) has maintained stable and productive scleractinian coral communities since the last large scale decreases in coral cover around 1999. The neighboring bank reef communities experienced the greatest losses to coral cover during this period and have yet to rebound (Lirman and Fong, 2007; Schutte et al., 2010) . Our an alysis of the past 16 - years of data from CREMP indicates that this trend has continued (Figure 2). We observed a greater percentage of live coral cover and greater coral diversity at five sit es within the inshore reef system spanning the lower and middle keys compared to five bank reef sites. Community level analysis of an external patch reef and offshore reef site, further support that scleractinian corals inhabiting inshore sites are not on ly more abundant (Figure 2) and more diverse (Figure 3), but mean colony size is significantly greater (Figure 7). Analysis of the 10 reefs from the CREMP indicated that the inshore patch reef system now accounts for greater than 70 % of the coral cover r emaining throughout the FKNMS regardless of the more consistent environment associated with offshore reefs. Bank reef habitats were characterized by lower SWTs along with a narrower range of thermal experience compared to inshore patch reefs (Figure 9). Additionally I identified significantly decreased levels of silicates at bank reef sites and a mean turbidity half that of inshore sites, providing support to previously reported trends of nutrients and turbidity in this area of the FKNMS (Lapointe et al., 2004) . Although the rapid loss of dominant acropori i d corals from the bank reef system caused a dramatic shift in reef biota (Patterson et al., 2002) as a consequence of the epizootic pathogen Serratia marsescens (Patterson et al., 2002; Sutherland et al., 2011) , this event alone does not explain the failure of the currently dominant coral species (Burman et al., 2012) to re - estab lish this zone over the past 20 - years given the apparent absence of other potential environmental constraints. 31 My analysis indicates that patch reefs and bank reefs of similar depth (6 m) within the middle and lower FKNMS support distinct scleractinian coral communities. The abundanc es of four species greatly influenced the ordination of inshore sites away from offshore sites; Acropora palmata , Siderastrea siderea , Montastraea cavernosa , and Porites astreoides (Figure 4). The influence of A. palmata was least revelatory as this known specialist inhabits a narrow habitat range including high relief and inner line spur and groove habitats found only in the bank reef zone (Miller et al., 2008) . The current endangered status of A. palmata Siderastrea siderea , the most common scleractinian coral currently inhabiting the FKNMS (Burman et al., 2012) , was highly prevalent regardless of the zone. B ecause the goal of this study was to identify sources of variation in the coral cover of inshore and offshore sites, the known generalist nature of S. siderea (Burman et al., 2012) precluded this species as well. The remaining two species, P. astreoides and M. cavernosa , although considered eurytopic , inhabited inshore and offshore zones with differing degrees of prevalence suggesting dissimilar tolerances to the str essors inherent to each zone. I selected these species as a proxy to identify site and species dependent factors that may contribute to the continued trend of decreased coral cover at offshore bank reefs compared to inshore patch reefs. Reciprocal transplantation of M. cavernosa and P. astreoides between Birthday Reef (inshore site) and Acer 24 (offshore site), revealed that fragments from both species transplanted to the inshore site, displayed significantly lower brightness in images than conspecifics and ramets transplanted to the offshore site regardless of the collection site (Figure 11). I interpret this result to signify decreased levels of stress at inshore reefs resulting in greater symbiont concentrations within the host or increased pigment concentration within the algal symbionts ( Symb iodinium spp. commonly referred to as zooxanthellae). Although increases in symbiont density can also occur when inorganic nitrogen concentrations increase (Marubini and Davies, 1996; Muscatine et al., 1989) , my study of the abiotic 32 differences between the patch reef and bank reef zone did not yield a signif icant difference in inorganic nitrogen forms between zones. Instead, inshore sites displayed increased seawater temperatures and a wider range of turbidities. Further, the two CREMP sites nearest to our transplant sites, Western Washer Women (inshore) an d Looe Key (offshore), displayed significantly different turbidities during the winter ( µ = 3.1 NTU and µ = 0.26 NTU respectively) and summer ( µ = 0.8218 NTU and µ = 0.480 NTU respectively). From this information differences in saturation and brightness o f transplanted coral colonies may be a function of the effects of turbidity and temperature. Temperature and turbidity have been identified as mediators of growth for Orbicella annularis populations n ear Key Largo, FL (Hudson, 1981) . While transplantation site affected the brightness of coral fragments, the conditions previously experienced by a transplanted fragment (Collection Site) did not have a significant effect on this measure of symbi ont response. Although this finding is by no means conclusive as to the nature of symbiont responses during this period, it provides evidence that the differences in stressors identified between these two sites were not significant enough to elicit a visi ble response. This finding is contradictory to studies that have reported significant interactions between the environmental history experienced by these two species and their response to future stress (Haslun et al ., 2011; Kenkel et al., 2011) . For instance, we have shown previously (Haslun et al., 2011) th at M. cavernosa from the Flower Garden Banks National Marine Sanctuary (FGBNMS), a well - developed and thermally stable scleractinian reef ecosystem, are more susceptible to increased thermal stress than conspecifics from the FKNMS, where increased SWTs are experienced. Similarly, P. astreoides has been shown to display experience dependent acclamatory responses via the expression of genes associated with calcification and metabolism when exposed to different temperature regime (Kenkel et al., 2013b) . Both of the aforementioned studies reported that pronounced differences in seawater temperature existed 33 between the collection locations. In this study, mean maximum and mean minimum temperatures for summer and winter respec tively, deviated by approximately 1 ° C between inshore and offshore zones, which is less than that observed between the FGBNMS and the FKNMS (Haslun et al., 2011) or the more extreme temperature regimes at near shore sites (< 1km from shore) of the FKNMS (Kenkel et al., 2013b) . It is possible that the dissimilarity in SWT observed between our sites was not acute enough to illicit an acclamatory effect in the observed brightness of P. astreoides and M. cavernosa . Bleaching (loss of symbionts) of corals during summer seasons is a common response to increased stress from light and temperature levels (Fitt et al., 2000) and not necessarily detrimental to the organism. caused by symbiont malfunction (Strychar et al., 2004) . Seasonal and widespread bleaching are hypothesized to be critical to the adaptation of corals in the face of the increasing magnitude and frequency of stressors (Buddemeier and Fautin, 1993; Fautin and Buddemeier, 2004; Guest et al., 2012) . Coral fragments experiencing the conditions at the inshore si te displayed seasonality in colony brightness, characterized by a summer stress period and a winter compensatory period (Figure 12). The seasonal pattern was also reflected in the significant relationship identified between temperature and brightness for these corals. Therefore, seasonal stress levels from temperature remain coupled to bleaching at Birthday reef and may be critical to the continued success of coral at inshore reefs. Seasonality in coral fragment brightness was not detected for fragments transplanted to Acer 24. On the contrary, we observed a significant positive linear trend between brightness and month, indicating that fragments of both species of coral became progressively lighter over this two - year period. Therefore, bleaching (the l oss of symbionts) and temperature stress can be considered uncoupled for P. astreoides and M. cavernosa experiencing the conditions at Acer 24. Given the narrower temperature range characteristic of this site, this result is intriguing. From our results we hypothesize that stress resulting from increased 34 irradiance (i.e. decreased turbidity), characteristic of offshore sites, provides an additional source of stress increasing the cumulative level during summer and winter periods beyond that of corals at i during the winter season resulting in a state of chronic bleaching. Chronic bleaching (an archetype of chronic distress) can be difficult to observe given its non - lethal nature (Lasker and Coffroth, 1998) . Horizontal linear extension was evident at both transplant sites throughout the course of our study (pers. obs.), however we were unable to determine whether or not the degree of growth differed between sites. In order to detect potential differences in fitness associated with chronic distress, we investigated site dependent community characteristics. Eventual mortality may occur when organisms must allocate resources to protection and maintenance (Lasker and Coffroth, 1998) rather than fecundity and growth. We observed a significant difference between the mean colony area of M. cavernosa and P. astreoides at Birthday reef and Acer 24, as well as another commonly observed species, S. siderea (a known generalist). Similar observations have been noted outside of the Florida Keys (Edmunds and Elahi, 2007) . Following dramatic decreases in the abundance of O. annularis (formerly Montastraea annularis ) at a reef near St. John Island in the United States Virgin Islands, the mean size of corals continued to decrease over a five - year period, from 1999 - 2003, despite the stabilization of perce nt coral cover (Edmunds and Elahi, 2007) . The authors projected that this species of coral would become locally extinct if the level of stressors remained unchanged over the next 30 - 50 years. Corals at offshore reefs of the FKNMS appear to be experiencing this trend now. 35 1.6 Conclusion Offshore bank reefs harbor relict communities of scleractinian corals and these communities do not show signs of rebounding. Although the stressors associated with this zone appear lower than those associated with more successful inshore patch reefs our r esults indicate that corals inhabiting offshore shallow reefs are experiencing more significant levels of stress compared to conspecifics from inshore patch reefs. This result may not be reflected in the acuteness of stress but rather the absence of a pha se to recover from seasonal stressors. In support of this claim we provide several tracts of evidence. First, the brightness of corals (an indication of symbiont density) transplanted to an offshore site became progressively lighter over a two - year perio d regardless of the site of collection while corals transplanted to an inshore site displayed seasonality in brightness. Second, the linear relationship between seawater temperature and brightness was significant for corals transplanted to inshore sites b ut this relationship did not exist for corals transplanted to an offshore site. Third, the mean colony size of the two species used for transplantation as well as a common generalist, were smaller at offshore sites. Finally, we identified a factor, turb idity that provides an explanation supporting the potential for chronic stress to occur at offshore sites. We propose that decreased turbidity results in increased irradiance at offshore sites resulting in greater cumulative stress than is present at insh ore reefs. The results presented in this study provide support for increased protection and awareness of reefs inshore to the bank reef habitat. It also provides important information regarding the important synergism between changing temperature and irr adiance experienced by corals. Increased turbidity provides both a refuge from increased irradiance as well as a potential food source to counteract decreases in algal carbon molecule production for the cnidarian host during periods of eutrophication as l ong a s sedimentation is not significant (B ongiorni et al., 2003) . 36 CHAPTER 2 LOCAL ADAPTATION OF PORITES ASTREOIDES BETWEEN INSHORE AND OFFSHORE METAPOPULATIONS INHA BITING THE FLORIDA R EEF TRACT 2.1 A bstract Dramatic changes to the coral communities of the Florida Reef Tract (FRT) have been observed over the past 30 years. Coral cover throughout this reef system is now disproportionately distributed with greater than 70% of the remaining coral cover accounted for within the inshore patch reef zone (< 2 km from shore) compared to 30% within the offshore bank reef zone (> 5 km from shore). Coral community changes along the FRT have been attributed to mortality stemming mainly from thermal stress and disease , al though other stressors undoubtedly contribute (e.g. irradiance) . Offshore patch ree fs, however, experience a smaller seawater temperature (SWT) range than inshore patch reefs (i.e. less thermal stress) suggesting that disease contributes more to declines in coral cover than SWT at offshore reefs . I examined the degree to which the immune system was activated in Porites astreoides originating from the inshore and offsh ore reef environments to determine if increased activation was associated with decreased coral cover . Colonies from a n inshore and offshore site were reciprocally transplanted and the expression of three genes determined biannually for two years (two summer, two winter periods). Variation in the expression of eukaryotic translation initiatio n factor 3, subunit H (eIF3H), an indicator of cellular stress in P. astreoides , did not follow patterns of SWT change indicating the contribution of other stressors to this response (e.g. irradiance , disease ). Greater expression of TNF receptor associate d factor 3 (TRAF3), a signaling protein of the inflammatory response activated following pathogen recognition , was observed among corals transplanted to or located within the offshore environment indicating that the offshore habitat stimulates the immune r esponse to a greater degree than the inshore habitat (p < 0.001). Corals collected from the offshore site upregulated the expression of adenylyl cyclase associated protein 2 (ACAP2), which decreases innate immune system inflammatory responses, indicating a counteractive adaptive response to increased stimulation of the 37 immune system. T he refore, the offshore P. astreoides population, which is characterized by smaller mean coral colony size and decreased colony abundance was also associated with an increase in the immune response and a local ly adaptive response indicating potential fitness tradeoffs. Activation of the immune respons e is metabolically costly . Therefore, increased immune system activation at offshore reefs is likely a contributing factor to c oral community dynamics and declines along the FRT. 38 2. 2 I ntroduction Over the past 30 years Porites astreoides has become increasingly abundant along the Florid a Reef Tract (FRT) surpassing the previously dominant Acropora spp. and Orbicella annularis (Lirman and Fong, 2007) . Commonly described as a generalist scleractinian coral, P. astreoides is ubiquitous throughout the FRT (Crabbe, 2009) . Lirman and Fong (2007) speculate that the change in species dominance is a response to clim atic and anthropogenic stressors. An increase in the frequency and severity of warming events in conjunction with the spread of coral disease is hypothesized to have driven these community level changes (Harvell et al., 2002) . Several autecological factors have been identified in P. astreoides that contribute to its increased survivorship. First, among shallow (< 2 m depth) nearshore reefs a green color morph is prevalent due to increased levels of a particular mycosporine - like amino acid, asterina - 330, that confers resistance to ultraviolet radiation (Gleason, 1993) . Second, in contrast to other coral species, P. astre oides , focuses breeding efforts during the milder spring temperatures and some colonies remain gravid throughout the year. This reproductive strategy may provide the host with ample time to resupply energy stores after reproduction and increase survival d uring high temperatures in summer (Chornesky and Peters, 1987) . Using gene expression methodologies Kenkel et al. (2011) showed that resistance to thermal stress in P. astreoides is primarily dependent upon the degree of prior exposure (Kenkel et al., 2013b) . Additionally, we have shown t hat the thermal stress response of Montastraea cavernosa is also dependent upon the experienced non - lethal thermal history of a colony (Haslun et al., 2011) . These two cases suggest that prior exposure to thermal stress may be an important factor affecting survivorship of coral species . Along the FRT, corals inhabiting inshore patch reefs experience greater summer and winter variation in seawater temperat ure (SWT) than is experienced by corals at offshore bank reefs and increased coral 3 9 cover is linked to increased SWT variation along the FRT (Soto et al., 2011) . Mean summer maximum and mean winter minimum SWTs at inshore reefs are at least 1°C more extreme than experience d offshore (Haslun et al., 2015 in review). Porites astreoides inhabiting inshore reefs can therefore be expected to have a smaller population relative to the offshore due to their exposure to increased levels of thermal stress as increased stress places c onstraints on fitness related traits. Contrary to this prediction, the abundance of P. astreoides colonies is considerably lower at offshore sites than in inshore sites. Moreover, the mean colony diameter of this species decreases from 16 cm at inshore r only been reported at inshore reefs (Lirman and Fong, 2007; Haslun et al., 2015 in review) . A high abundance of small P. astreoides colonies (< 10 cm diameter) along this gradient indicates efficient recruitm ent at both sites whilst the sc arcity of large colonies (> 30 cm diameter) offshore is indicative of an increased potential for mortality and stress offshore. While gene expression studies suggest that P. astreoides is resistant to thermal stress (Kenkel et al., 2011) , and there is lower incidence of colony bleaching and increased colony growth during warmer SWTs (Kenkel et al., 2013a) , these o bservations do not adequate ly explain differences in abundan ce and colony size between inshore and offshore reefs in this study. In addition to increased temperature stress, microbial stress and disease have severely impacted FRT offshore bank reefs. Acropora cerviconis a nd A. palmata have been functionally types I, II, and III (Richardson et al., 1998) , white band disease types I and II (Ritchie and Smith, 1998) and white pox (Patterson et al., 2002) . Yellow band disease, and dark spots disease are also common throughout the Caribbean and impact many corals including P. astreoides (Cervino et al., 2001) . The spread of these diseases has been linked to increased summer temperatures, while colder winter temperatures tend to diminish disease spread and reduce coral mortality by decreasing bacterial numbers a nd pathogenicity (Bruno et al., 2007; Cervino et al., 2004; Ward, Jessica, Kiho, Kim, Harvell, 40 2007) . Therefore lower winter temperatures effectively decrease the activation of the host immune response to bioti c stress. The milder SWTs of offsho re bank reefs likely exacerbate disease related stress by failing to reduce the abundance of po tential activators of disease during win ter (e.g. microbe abundance a nd pathogenicity). Therefore I would expect to observe amplified immune activation in corals experiencing the offshore reef where warmer winter temperatures constrain pathogenic microbe activity to a lesser degree . Moreover, i ncreased activation of the innate immune system has been shown to decrease invertebrate fitness (Armitage et al., 2003; Sheldon and Verhulst, 1996) . O ffshore ree fs display both decreased coral colony size and decreased coral abundance (Haslun et al., 2015 in review; Lirman and Fong , 2007) resulting in decreased fecundity for P. astreoides ( Chornesky and Peters, 1987 ) . In this study, I reciprocally transplanted P. astreoides colonies from an inshore patch reef and offshore bank reef monitor ed the expression of three host - specific genes. Eukaryotic translation initiation factor 3, su bunit H (eIF3H) provided an indication of a general stress response whereas TNF receptor associated factor 3 (TRAF3) and adenylyl cyclase associated protein 2 (ACAP2) provided indications of activation of the immune system biannually (summer and winter) ov er a duration of two years. In doing so, we determi ned (1) the effect of the site - dependent environme nt on gene expression, (2) the influence of SWT on gene expression in P. astreoides t ransplanted to each site and (3), we examined if inshore and offshore populations displayed adaptive responses to each environment . 41 2.3 M ethods - - offshore bank reef environment, respectively (Figure 1 3 ). The reefs are located adjacent to one another along Hawk Channel within the lower region of the Florida Keys National Marine Sanctuary (FKN MS) and at similar depth s (6 m). Birthday reef is charact erized by greater abundance and diversity of scleratinian coral, increased variance in SWTs (including greater minimum and maximum temperatures during the winter and summer respectively) , and greater turbidity than Acer 24 (Haslun et al., 2015 in review ). 42 Figure 1 3 : The Lower Region of the Florida Keys is Presented Along with Several Orientating L ocations . The locations of each sample site are indicated by symbols. The inshore patch reef Birthday Reef is represented by a square and the offshore bank reef Acer 24 reef by a triangle. 43 2.3.1 Collection and t ransplantation Collection and transplantation of coral in this study was conducted under NOAA National Marine San ctuaries Permit # FKNMS - 2011 - 10. Porites astreoides fragments from Birthday and Acer 24 reefs were reciprocally transplanted. Fragments (16 cm 2 x 16 cm 2 ) of P. astreoides colonies (n = 6) were collected (from each site at a depth of 6 m by divers using SCUBA. Fragments were removed from a colony using a cold chisel and mallet. Following collection, fragments were transported by boat to the Mote Marine Laboratory Tropical Research Labor atory (MML TRL) in a cooler containing site - derived water. Each fragment was ha lved with an electric b rick - saw that was cooled and lubricated with sterile seawater. The resulting coral samples were placed in a shaded flow - through seawater syste m and equilibrated for 24 h. Following the recovery period, fragments were affixed to 6 cm x 6 cm concrete and a ragonite (3:1) bricks with All - Fix epoxy (Cir - Cut: Lafayette Hill, PA). Coral fragments were then allowed an additional 48 h to equilibrate prior to being returned to the site of origin or transplanted to alternate site (Haslun et al., 2015). Each fragme nt was sub - sampled bi - annually for two years during the summer (August - September) and winter (January February) by divers equipped wit h SCUBA. Sub - samples ( 2 x 2 cm ) were removed with a hack saw equipped with a tungsten carbide blade (Milwaukee CPO: P asadena, CA) and a cold chisel and mallet. This method prevented dislodging of fragments from the study site concrete blocks. On - board the ship, fragments were rinsed with sterile sea water, wrapped in combusted tin - foil, and then flash frozen in liquid nitrogen (LN 2 ). The entire process from collection to processing lasted less than 5 min for any single sample. Fixed samples were shipped to Michigan State University (East Lansing, MI) or the Annis Water Resources Institute (Muskegon, MI) on dry ice and stored at - 80°C prior to RNA extraction . 44 2.3.2 Sample processing and RNA i solation Coral fragments were first pulverized using a stainless steel dounce - style homogenizer chilled with LN 2 to retain sample integrity. Excess skeletal fragments were remov ed with forceps and the remaining sample further pulverized with a ceramic mortar and pestle in a shallow pool of LN 2 within the mortar . Mortar and pestles were cleaned with Alconox (Alconox Inc.: White Plains, NY), rinsed three times with de - ionized wate r, treated with RNase (RNase zap: Sigma - A ldrich, St. Louis, MO), and rinsed with ultra - pure deionized water (E - Pure; Thermo Fisher Scientific Inc.) between samples. Sample powder was transferred to a microcentrifuge tube and stored at - 80°C prior to RNA i solation. RNA was isolated from 110 mg of processed sample powder using a mixture of guanidine thiocyanate and phenol in a monophase solution (TRI Reagent: Sigma - Aldrich, St. Louis, MO). Materials used for weighing were chilled in LN 2 and the spatula treated for RNase as p reviously described. TRI Reagent (1 mL) was added to the sample and cell disruption facilitated via pipet aspiration . The sample was incubated for 10 min at room temperature and centrifuged ( 10,000 rcf, 10 min , 4°C ) to achieve phase separation . The aqueous phase was then transferred to a new tube with 250 µL of a 0.8 M sodium citrate and 1.2 M sodium chloride solution and shaken vigorously for 5 sec to rem ove polysaccharide contaminants. Isopropanol (neat, 250 µL ) was added and the microcentrifuge tube shaken vigorously for 15 sec. Samples in isopropanol were incubated (room temperature 10 min) and centrifuged (10,000 rcf, 10 min, 4°C ) to pellet the RNA. The RNA pellet was washed twice with 75% ethanol and centrif uged (7,500 rcf, 5 min , 4°C ). The resulting RNA extract was dissolved in RNase and DNase - free water (100 µL) and evaluated for RNA integrity with a Caliper Lab Chip GX (Perkin Elmer: Waltham, MA). RNA quality scores (RQS) greater than 6 were deemed of suf ficient quality for two - step reverse transcription quantitative real - time polymerase chain reaction (qRT - PCR) (Fleige and Pfaffl, 2006) . RNA extraction yielded approximately 2 µg of RNA. 45 2.3.3 Two - s tep qRT - PCR Isolated RNA ( 300 ng ) was treated with 1 unit of DNase 1 (Life Technologies: Waltham, MA) in DNase treated RNA (260 ng) was reverse transcribed with Superscript III first strand synthesis supermix (Life Technologies: Waltham, MA). Reverse transcription reactions were carr ied out in a 96 - well plate using an Eppendorf Mastercycler and the following thermal profile : 10 mi n 25°C, 30 min 50°C, 5 min 85°C. After denaturation of the enzyme at 85°C, RNase H (1 µL) was added to each reaction and incubated (37°C, 5 min ) to degrade the remaining template RNA. Reverse transcribed cDNA was purified from the reaction via precipitation. T o remove potential inhibitors to qRT - PCR , 7.5 M ammonium acetate (Sigma Aldrich: St. Louis, MO) ( 6 µL ) was added followed by - 20°C i sopropanol (neat, 50 µL). The sample was inverted 10 times to mix and chilled ( - 80°C, 1 h ) to precipitate the cDNA. Precipitated samples were centrifuged (18,000 rcf, 15 min ) to pellet the cDNA. The precipitation reagents were decanted and pellet washed twice with 75% ethanol (1 mL) . Washed cDNA was centrifuged (18,000 rcf, 15 min). T he pellet was allowed to air dry (room temperature, 10 - 15 min ) and re - suspended in ultra - pure water (104 µL) to yield a final cDNA concentration of 2.5 ng µL - 1 . cDNA was stored at - 20°C for less than 1 week prior to qRT - PCR. 2.3.4 qRT - PCR v alidation Sequences of transcripts of interest were obtained from the P. astreoides SymBioSys database ( http://sequoia.ucmerced.edu/SymBioSys/ ). Primers were created with Primer3 (Rozen and Skaletsky, 1998) software applying the selection criteria outlined in Table 1. The top scoring primer pair was selected for qRT - PCR validation. A standard curve relating the threshold cycle (Cq) to the l og concentration of cDNA along a 2 - fold serial dilution was used to validate primers ( 5 ng µL - 1 , 2.5 ng µL - 1 , 46 1.25 ng µL - 1 , 0.625 ng µL - 1 , and 0.3715 ng µL - 1 ) . Primer pairs that amplif ied a single product according to d issociation curves, displaying a hig hly linear relationship between Cq and concentration (R 2 > 0.99) and adequate amplification efficiency (3.0 < E < 3.6), were considered valid for analysis (Table 2). Real - time PC R reactions were 10 µL in volume (1 µL template cDNA, 5 µL Power SYBR green master mix (Life Technol ogies: Waltham, MA), 1.5 µL primer pair (f inal concentration 250 nM), 2.5 µL DNase and RNase - free water ) . Amplification and detecti on of transcripts was performed on an Applied Biosystems 7900HT real - time PCR system in 384 - well pla To prevent potential run - to - run variation and the need for inter - run calibrator samples, all samples were run in duplicate on a single 384 - well plate for each gene of interest (Derveaux et al., 2010) . Table 1 : Selection Criteria for Primers Used to Amplify Transcripts for Quantitative Real - Time Polymerase Chain R eaction. Primer Selection Parameter Criteria Product size 50 - 150 Number of results returned 5 Max repeat mispriming 12 Max template mispriming 12 9 Pair max repeat mispriming 24 Pair max template mispriming 24 Primer size 18 < 20 > 22 Primer Tm 57 < 59 > 61 Max Tm difference 1 Primer GC % 20 < 50 > 80 Max self - complementarity 2 Max 0 First base index 1 - complementarity 3 Max poly - X 4 47 Table 2: The Amplification Efficiency, Primer Sequences of Each Gene of Interest, and Control G enes are Presented in the T able. Genes of Interest Abbreviation Primer Sequence - Efficiency TNF receptor - associated factor 3 TRAF3 F: GTCTGGCTCCTCCCATCTTT R: GCCTCCAGCATTCTAACCTG 2.03 Adenylate cyclase associated protein 2 ACAP2 F: TCGTCTGGAGTCTGCTGCT R: TCTGCCACTTTGCCGTTTA 2.04 Eukaryotic initiation factor 3, subunit H EIF3H F: TTGATTGATACCAGCCCACA R: ACAAACTGCTTTGCTTTCCC 1.97 Control Genes 60S ribosomal protein L11 RPL11 F: TTTCAAGCCCTTCTCCAAGA R: GACCCGTGCTGCTAAAGTTC 1.94 Cathepsin L CATL F: GGAAGGATTACTGGCTGGTC R: GGATAGATGGCGTTTGTGG 2 2.3.5 qRT - PCR a nalysis A Bayesian model - based approach (Matz et al., 2013) was applied to analyze qRT - PCR Cq data. Raw Cq values were transformed to molecular counts creating values with a linear rather than exponential relationship. A Mar kovian Chain Monte Carlo generalized linear mixed model (MCMCglmm) with Poisson log - normal distribution was then applied. Applying an MCMC - based approach had several advantages over the traditional delta delta Cq methodology for this study (Livak and Schmittgen, 2001) . First, it is rare that control genes behave perfectly stable in a natural setting. The MCMC - based approach allows for modeling of the variance associated with control genes and builds this error into the predictions. Secondly, this approach allows for the addit ion of random error terms that allow and correct for unequal template loading, resulting in normalization of the data. Thirdly, the hierarchical model produced allows for simultaneous determination of the treatment effects across all genes of 48 interest rel ative to control genes improving upon the gene by gene analysis applied with the delta delta Cq method (Matz et al., 2013) . I s eparated the analysis into two models a priori to identify transplantation site - dependent effects (Equation 1) and collection site - dependent effec ts (Equation 2). Molecular counts were the single gene gene expression , Tran represented coral fragments grouped by site of transplant CollectionSite represented co ral fragments grouped by site of origin , and SeasonYear combination ( i.e. Summer 2012, Winter 2012). Conditions in brackets indicate random error terms and include the sample specific error, the gene specific sample error, and the gene specific error in the order presented in each model. Equation 1: Equation 2: Model parameters included 15 , 000 iterations, a thinning interval of 10, and sample size of 1 , 000. A less - informative inverse Wishart prior with assumed variance of 1 and the degree of belief parameter at 0 was used for calculating variance components of all non - control genes. Control genes were allowed to vary on average 1.2 fold across the explanato ry variables. Credible intervals (95% posterior probabilities) for fixed factors were calculated based on MCMC sampling. 49 2.3. 6 Genes of i nterest 2.3. 6 .1 Tumor necrosis factor receptor associated f actor 3 (TRAF3) The protein TNF receptor assoc iated factor 3 (TRAF3) perpetuates the activation of immune responses following host detection of bacterial and viral pathogen associated molecular patterns (PAMPs) in the surrounding environment (Bagchi et al., 2007; Häcker et al., 2011 ; Medzhitov and Janeway Jr., 2002; Rowley and Powell, 2007) . Therefore, expression of TRAF3 provides an indica tion of PAMP detection and the resulting response . 2.3.6.2 Adenylate cyclase associated p rotein 2 (ACAP2) Adenylate cyclase associated proteins (ACAPs) are activated to prevent over - activation of the inflammatory response (Montminy, 1997; Serezani et al., 2008; Shima et al., 2000, 1997) . Therefore ACAP2 reduces responses of the immune system that may be detrimental to the host . 2.3.6. 3 Eukaryotic t ranslation Initiation Factor 3, subunit H (eIF3H) Eukaryotic translation initiation factors (eIFs) regulate the translation of cytoplasmic mRNAs and degradation of proteins (Zhang et al., 2008) . Eukaryotic translation initiation factor 3, subunit H (eIF3H) is a component of the translation initiation complex that is upregulated in P. astreoides during periods of increased SWT (Matz, 2013) . Therefore this gene is included as an indicator of thermal stress. 50 2.4 Results 2.4.1 Site temperature r egime The two sites displayed similar trends in SWT change as a function of season reflecting their close proximity (Figure 14 ). Seawater temperatures at Acer 24 remained lower during the summer and greater during the winter compared to those observed at Birthday reef (Table 3) . During winter lows and summer highs, SWT differed by 0.5 to 1°C between the sites. Additionally, the frequency of mean daily temperatures above 30°C was greater at Bir thday than Acer 24 reef. The preceding 8 days to sampling during the winter of 2012, seawater tempe rature increased significantly at Birthday reef ( 3°C increase) and Acer 24 ( 1°C increase) (p = 0.0024 and p = 0.037 respectively; Table 3). Conversely, during the winter of 2013 a significant decrease in temperature wa s observed the 8 days preceding sampling at Birthday reef ( 3°C decrease) and Acer 24 (2°C decrease) (p = 0.001 and p = 0.000 respectively; Table 3). The magnitude of the temperature increase during the winter of 2013 was greater than that which occurred during the winter of 2012. There were no significant changes in temperature during the summer months over the week preceding sampling. 51 Figure 14: The Figure Displays Hourly Water Temperature for Birthday Reef (Inshore Patch Reef) and Acer 24 (Offshore Bank Reef) Over the Course of the Two - Year Reciprocal Transplantation E xperiment. Daily water temperature means are presented for the day of sampling for each of the four sample periods as well as the mean daily water temperatures for the pr evious 6 days prior to sampling corals at the Acer 24 and Birthday Reef field site . Table 3: Seawater Temperatures Reported at the Date of Collection at the Offshore Site (Acer 24 Reef) and the Inshore Site (B irthday Reef) . The slope of temperature chan ge during the week preceding sampling and p - value associated with the linear regression are also displayed. Field Site Date (Year - Month) SWT at Collection (°C) One - week SWT Slope Regression p - value Acer24 Reef Winter 2012 24.5 0.06592 0.0377 * Summer 2012 30.6 - 0.08914 0.418 Winter 2013 22.9 - 0.49137 0.000467 *** Summer 2013 30.0 - 0.01571 0.513 52 Birthday Reef Winter 2012 25.2 0.19554 0.0024 ** Summer 2012 31.5 0.04378 0.244 Winter 2013 22.0 - 0.59509 0.00115 ** Summer 2013 30.3 - 0.05640 0.0765 . 2.4.2 Effects of site and season on pooled GOI t ra nscript a bundance Fragments originating from Acer 24 exhibited greater transcript abundance across all GOI than those originating from Birthday reef (p = 0.010 ) . This indica ted a potential effect of site of origin on gene expression. Additional support for the site of origin influencing a fragments response was provided by principal coordinate analysis (PCoA) of the Manhattan distances of transcript ab und ances among the GOI (Figure 15 ). Samples formed clust ers dependent on site of origin rather than the alternate transplant site . The greatest dissimilarity between treatments was between fragments originating from Acer 24 and transplanted to B irthday reef and those originating from Birt hday reef and transplanted back to Birthday reef. The site a fra gment was transplanted to did not a ffect transcript abundance when GOI were collectively analyzed. 53 Figure 15: Principal Coordinate Analysis Results Indicating a Collection Site - Dependent Effect on Transcript A bundance. Manhattan distances of transcript abundances were used to create the distance matrix. Axis 1 and 2 are plotted. The labels represent the mean of a xis 1 and axis 2 for each group. Grey points indicate corals collected from Acer 24 reef and black points represent those collected from Birthday reef. A significant linear relationship was observed between the slope of SWT change associated with the 8 da ys preceding sampling and the second PCoA axis (p = 0.005) while the temperature at the time of sampling was not correlated to either axis (Table 2). This observation indicates that SWT change contributed to the difference in expression be tween transplant treatments along axis 2 (6%) , however a large majority of the variance (62%) was not associated with SWT change during the preceding week . Transcript abundance differed between the winter and summer of 2013 for both the collection site (p = 0.012) and tra nsplantation site (p = 0.008) models. Transcript abundances were lower during the summer of 2013 than the winter of 2013. 54 2.4.3 Gene specific responses of three genes of i nterest 2.4.3.1 TRAF3 expression Porites astreoides fragments transplanted to Acer 2 4 reef displayed significantly greater transcript abundance than fragments transplanted to Birthday reef (p = 0.01 ; Figure 16 , Table 4 ). This difference was driven by increased transcript abundances during the summers of 2012 and 2013 at Acer 24 reef comp ared to Birthday reef (p < 0.001 for both comparisons ; Figure 16 , Table 4 ). During the winter of 2012 TRAF3 gene expression was similar between the two reefs and reached comparable levels to those observed during each summer. During the Acer 24 sampling periods, transcript abundances observed during the winter were less than those quantified the following summer, indicating a potential effect of season on TRAF3 expression at Acer 24. This pattern was not observed at Birthday reef. Instead, a significant difference in transcript abundance was only evident between the winter of 2012 and the summer of 2013 (p < 0.001 ; Figure 16 , Table 4 ). 55 Figure 16: The log2 Scaled Abundances of TRAF3 Transcripts from Corals Reciprocally Transplanted Between an Inshore Site (Birthday Reef) and an Offshore S ite (Acer 24 Reef ). The left panel displays fragments grouped by the site of origin independent of the site transplanted to and the right panel displays fragments grouped by the site transplanted to independen t of the site of origin. Samples were collected for analysis during the winter (February) and summer (September) of 2012 and 2013. Points represent the posterior means following MCMC generalized linear mixed modeling. Error bars indicate 95 % credible in tervals defined by the model. Table 4: Two - Way Factorial Mixed Effects Model for T ranspl ant Site - Dependent and Seasonal Effects on Transcript A bundance of Porites astreoides Following Reciprocal Transplantation Between an Inshore Patch Reef (Birthday Ree f) and Offshore Bank R eef (Acer 24 Reef ). The results for the three genes of interest adenylate cyclase associated protein 2 (ACAP2), eukaryotic initiation factor 3 subunit H (EIF3H), and TNF receptor associated factor 3 (TRAF3) are presented. Comparison s have been made to transcript abundances of samples transplanted to Acer 24 reef and sampled during the winter of 2013. GOI Comparison Posterior Mean Lower 95% CI Upper 95% CI pMCMC ACAP2 Birthday - 0.435 - 0.937 0.029 0.086 Winter 2012 - 0.03757 - 0.49681 0.44522 0.926 Summer 2012 0.019 - 0.372 0.500 0.938 56 Summer 2013 - 0.14493 - 0.61418 0.36624 0.576 Birthday: Winter 2012 0.49994 - 0.21072 1.17273 0.132 Birthday: Summer 2012 0.40865 - 0.24205 1.04486 0.210 Birthday: Summer 2013 - 0.30511 - 0.86000 0.25533 0.312 EIF3H Birthday - 0.208 - 0.637 - 0.173 0.342 Winter 2012 - 0.08121 - 0.50590 0.24429 0.700 Summer 2012 0.34064 - 0.02892 0.74230 0.076 . Summer 2013 - 0.14893 - 0.59086 0.29031 0.508 Birthday: Winter 2012 0.06488 - 0.42012 0.68499 0.828 Birthday: Summer 2012 - 0.30511 - 0.86000 0.25533 0.312 Birthday: Summer 2013 1.00556 0.43073 1.65886 <0.001 *** TRAF3 Birthday 0.76547 0.15549 1.40063 0.010 * Winter 2012 1.37740 0.73687 2.03280 <0.001 *** Summer 2012 1.80851 1.19198 2.48163 <0.001 *** Summer 2013 1.20155 0.50582 1.96071 <0.001 *** Birthday: Winter 2012 - 0.60505 - 1.45238 0.40027 0.182 Birthday: Summer 2012 - 1.21753 - 2.07043 - 0.28946 0.008 ** Birthday: Summer 2013 - 1.81851 - 2.85665 - 0.86877 <0.001 *** 57 Although site of origin did not impact the expression of TRAF3 (Table 5) , differences between sampling periods were observed (Figure 16) . The transcript abundance observed during the winter of 2013 was significantly lower than the expression observed during both the winter and summer of 2012 (p < 0.001 for each comparison). During a g iven sampling period site of origin did not affect TRAF3 expression, unlike that observed for the site a coral was transplanted to. Table 5: Two - Way Factorial Mixed Effects Model for Collection Site - Dependent and Seasonal Effects on Transcript A bundance of Porites astreoides Following Reciprocal Transplantation Between an Inshore Patch Reef (Birthday R eef) and Offshore Bank R eef (Acer 24 Reef ). The results for the three genes of interest adenylate cyclase associated protein 2 (ACAP2), eukaryotic initiation factor 3 subunit H (EIF3H), and TNF receptor associated factor 3 (TRAF3) are presented. Comparisons have been made to transcript abundances of samples transplanted to Acer 24 reef and sampled during the winter of 2013. GOI Comparison Posterior Mean Lower 95% CI Upper 95% CI pMCMC ACAP2 Birthday - 1.147154 - 1.670249 - 0.666332 <0.001 *** Winter 2012 - 0.013090 - 0.446707 0.396193 0.958 Summer 2012 0.277053 - 0.157765 0.713639 0.200 Summer 2013 0.119924 - 0.364999 0.552681 0.618 Birthday: Winter 2012 0.394370 - 0.243777 1.033281 0.234 Birthday: Summer 2012 - 0.193949 - 0.824670 0.472759 0.582 Birthday: Summer 2013 0.351856 - 0.372941 1.128164 0.374 EIF3H Birthday - 0.586846 - 1.032692 - 0.112198 0.018 * Winter 2012 - 0.266735 - 0.671877 0.103723 0.184 Summer 2012 - 0.002953 - 0.389189 0.347585 1.000 Summer 2013 0.082943 - 0.337972 0.471331 0.678 Birthday: Winter 2012 0.488955 - 0.150934 1.031597 0.108 58 Birthday: Summer 2012 0.460195 - 0.172337 0.984013 0.124 Birthday: Summer 2013 0.839239 0.196500 1.522053 0.022 * TRAF3 Birthday 0.362722 - 0.420370 1.282007 0.394 Winter 2012 1.473018 0.761331 2.084224 <0.001 *** Summer 2012 1.666734 0.975813 2.272533 <0.001 *** Summer 2013 0.614901 - 0.094805 1.261963 0.086 . Birthday: Winter 2012 - 0.852657 - 1.759411 0.160360 0.086 . Birthday: Summer 2012 - 0.985686 - 1.978128 - 0.025191 0.054 . Birthday: Summer 2013 - 0.930772 - 1.904549 0.089161 0.072 . 2.4.3.2 e IF3H expression The expression o f e IF3H was significantl y greater among corals originating from Acer 24 compared to Birthday reef site (p = 0.018; Table 5). This result was apparent despite t he similarity in expression of e IF3H observed for all but one of the sampling periods, winter 2013 (p < 0.022; Figure 17 , Table 5 ). The site a coral frag ment was transplanted to significan tly affected the expression of e IF3H during summer months but not winter months (Figure 17 ). During the summer of 2012 expression was greater for corals transplanted to Acer 24 compared to Birthday reef (p < 0.001) but t his significant trend was reversed the following summer (p < 0.001). 59 Figure 17: The log2 Scaled Abundances of eIF3H Transcripts from Corals Reciprocally Transplanted Between an Inshore Site (Birthday Reef) and an Offshore S ite (Acer 24 Reef ). The left panel displays fragments grouped by the site of origin independent of the site transplanted to and the right panel displays fragments grouped by the site transplanted to independent of the site of origin. Samples were collected for analysis durin g the winter (February) and summer (September) of 2012 and 2013. Points represent the posterior means following MCMC generalized linear mixed modeling. Error bars indicate 95 % credible intervals defined by the model. 2.4.3.3 ACAP2 expre ssion Transcript abundances of ACAP2 were sign ificantly affected by site of origin (p = 0.0 00; Table 5). Corals from Acer 24 displayed greater ACAP2 expression for each sampling period compared to corals from Birthday reef (Figure 18 ). This site of origin - dependent effec t on transcript abundance was not influenced by sampling period. Additionally, the site that a coral fragment was transplanted to did not affect ACAP2 transcript abundance. 60 Figure 18: The log2 Scaled Abundances of ACAP2 Transcripts from Corals Reciproc ally Transplanted Between an Inshore Site (Birthday Reef) and an Offshore S ite (Acer 24 Reef ). The left panel displays fragments grouped by the site of origin independent of the site transplanted to and the right panel displays fragments grouped by the si te transplanted to independent of the site of origin. Samples were collected for analysis during the winter (February) and summer (September) of 2012 and 2013. Points represent the posterior means following MCMC generalized linear mixed modeling. Error b ars indicate 95 % credible intervals defined by the model. 2.4. 4 Summary of factors affecting host gene e xpression in P. astreoides Distinct patterns of transcript abundance were identified for each of the three genes of interest (GOI). The expression of TRAF3 differed between winter and summer but only for samples transplanted to Acer 24. Significant transplant site - dependent effects were apparent in the expression of e IF3H d uring summer while a site of origin - dependent effect was observed for ACAP2. W e also observed that the direction and magnitude of the slope associated with the previous week of temperature change was a 61 significant factor affecting gene expression while the previous days SWT during a given sampling event was not. 62 2.5 D iscussion Along the FRT corals inhabiting offshore bank reefs display decreased growth and abundance (e.g. fitness) compared to corals inhabiting inshore patch reefs (Haslun et al., 2015). Although elevated SWTs are generally associated with incre ased coral stress , higher SWTs and increased temperature variation are characteristic of inshore patch reefs to a greater extent than in the offshore (Soto et al., 2011) . An alter native ex planation for decreased offshore coral abundance is that SWT variation affects the activation of the immune system by influencing the level of biotic stress experienc ed by coral inhabitants. Increased temperature variability will control pathogen prevalence while decreased variability presents less stressful growth conditions (Harvell et al., 2002; Price and Sowers, 2004) . I determined if the expression of genes associated with the activation of the immune system were environment - dependent and if adapt ive responses to environment - dependent biotic stress had occurred. We identified increased activation of the immune system among all corals transplanted to the offshore site as well as an adaptive response to activation of the immune system in corals orig inating from the offshore site. The role of each gene examined will be discussed in turn to describe how SWT indirectly influences biotic stress level contributing to the observed differences in growth and abundance between the inshore and offshore sites. 2.5.1 Activation of host coral immune p athways: TNF receptor associated factor 3 expression The protein TNF receptor associated factor 3 regulates the activation of the innate immune response along the MyD88 dependent and TRIF dependent pathways (Bagchi et al., 2007; Häcker et al., 2011) . Following activation of TLRs by PAMPs, TRAF3 activates the production of effector molecules comprising the aforementioned pathways of the immune system, resulting in inflammatory responses ( Häcker et al., 2011) and viral molecular patterns in the surrounding environment. My results indicate that it is not the site of 63 origin that controls TRAF3 expr ession but rather the environmental conditions a coral host is currently exposed to. For this particular gene, a population dependent difference would be unlikely because toll - like receptors are conserved across animal phyla and activation occurs followin g the recognition of general rather than specific PAMPs (i.e. all bacterial lipopolysaccharides vs. species specific variants). The general and conserved nature of this pathway likely constrains selection on TLRs. The effect of local environment on TRAF3 expression was observed insofar as Porites astreoides transplanted to the offshore site displayed greater TRAF3 expression than those transplanted inshore in summer (p < 0.001). Increased SWT is often directly linked to responsive physiological changes i n corals. Contrary to this generalization, increased temperatures during the summer did not co - occur with increased TRAF3 activation. Instead, mean daily temperatures at the inshore site were on average 1°C less than those of the offshore site. While th e second principal coordinate axis, which delineated differences in gene expression among treatments, displayed a significant relationship with temperature variation the week prior to sample collection (p < 0.005, R 2 = 0.136), separation between treatment groups along this axis was minimal. Greater variation between transplants was identified along the first principal coordinate and a relationship with SWT was not observed (Figure 14 ). Therefore environmental factors other than SWT must contribute to vari ation in TRAF3 expression along this primary axis. During the winter, however, increased TRAF3 expression (p > 0.000) was observed following a period of increasing SWT whereas the lowest TRAF3 expression was observed following a period of decreasing SWT ( Table 3) . Thus cooling SWT during the winter, which reflect the inshore site SWT, was associated with less TRAF3 expression whereas warming SWTs during the winter enhanced TRAF3 expression, a characteristic of the offshore site. The direct link between T RAF3 expression and the recognition of microbial derived molecular patterns through TLRs indicates that biotic stress may be an additional contributing factor in this environment - 64 dependent effect. However, during winter periods, high SWT undoubtedly contr ibutes to the level of biotic stress experienced (Strychar and Sammarco, 2010; Strychar, 201 4 ). Bacterial abundance decreases du ring the winter but shorter an d warmer winter seasons diminish this effect (Harvell et al., 2002) . Because warmer SWTs increase the metabolic and growth rates of microorganisms (Price and Sowers, 2004) many diseases affecting marine macro - organisms increase in prevalence including those affecting corals (Bruno et al., 2007; Cerrano et al., 2000; Sussman et al., 2003) . Not surprisingly, seasonal variation in temperature (i.e. winter vs . summer) has been id entified as one of the most prominent factors controlling the rate of microbial growth (Fuhrman et al., 2015; Gilbert et al., 2012; Jiang and Paul, 1994) . Therefore during warmer periods (summer), an in creased abundance of foreign substances capable of binding TLRs is expected as a result of increase proliferation and pathogenicity of microorganisms. Conversely, cooler periods are expected to result in decreased biotic stress and subsequent TRAF3 expres sion. We identified both of these trends in TRAF3 expression: decreasing winter temperatures limited TRAF3 expression vs warm periods consistent with the greatest levels of TRAF3 expression. Moreover, on an annual basis, temperature variation is greater at the offshore compared to the inshore site. Therefore according to the hypothesis laid out by Harvell et al. (2002) and evidence supplied by others (Cerrano et al., 2000; Price and Sowers, 2004; Sussman et al., 2003) , however, the o ffshore sites warmer winter SWTs, fails to explain the accumulation of TLR compatible molecular patterns and the resulting activation of the immune system in the offshore relative to the inshore. Additionally, exposure of the coral fragments from this stu dy to increased temperature (32°C) and lipopolysaccharide (5 µg mL - 1 ) indicated that corals originating from the offshore site activated the immune system to a greater degree than the inshore site (Haslun et al., in review ). Corals transplanted to the off shore site likely encounter more immune activating compounds and therefore displayed significant upregulation of TRAF3 relative to a control treatment (28° C). C orals originating from the inshore site displayed expression similar to the control and signifi cantl y lower than that of corals with an 65 offshore origin . Therefore corals that had experienced increased immune system activation inherent to the offshore site upregulated TRAF3, whilst those from the inshore site did not activate this response as greatl y. Upregulation likely reflects increased pathogen expo sure from the local environment (i.e. offshore). The differential TRAF3 expression I observed in corals transplanted to inshore and offshore sites indicates local differences in the degree of immun e system activation. Moreover, our results indicate that the degree of immune ac tivation is linked to SWT variation of a site. Lower winter SWT decreased immune system activation while the milder temperature regime of the offshore site increased activati on. These temperature changes are undoubtedly linked to the abundance of PAMPs capable of activating the immune response although other abiotic factors such as irradiance and nutrients not quantified here likely contribute as well . Identification of the concentrations of immune system stimulating compounds present at inshore and offshore reefs is required to fully support this conclusion, however, increased expression of the immune system following exposure to the synergistic effect of increased temperatu re and LPS has been observed in offshore P. astreoides relative to inshore, indicating increased exposure to PAMPs offshore. Climate warming is expected to increase winter minimum temperatures along coastal regions like the FRT and result in greater insho re activation of the immune system. This will further exacerbating the currently observed activation of the immune system in the offshore environment. 2.5.2 Cellular stress response: eukaryotic translation initiation factor 3, subunit H (e IF3H) Eukaryotic translation initiation factor 3, subunit H (eIF3H) is a component of the translation initiation complex formed by eIFs and therefore contributes to the synthesis rather than degradation of proteins (Zhang et al., 2008) . This g ene is upregulated in P. astreoides during periods of stress (Matz et al., 2013) . 66 Upregulation of eIFs results in increased protein production that can counteract intracellular stress following metabolic dysfunction (Muñoz and Castellano, 2012) as observed in yeast (Singh et al., 2013) . Increased expression of eIF3H is therefore an indication of stress. Although we o bserved differential expression of eIF3H among corals transplanted to inshore and offshore sites during the summer, a consistent site - dependent effect on expression between summers was not observed. In fact, during 2012, eIF3H expression was greatest in c orals transplanted to the inshore site, whilst during the following year corals transplanted to the offshore site displayed the greatest expression. Offshore SWT varied between 29°C and 30°C prior to sampling for gene expression during the summer of 2012 while SWT at the inshore site increased from 28°C to 32°C during the same period (Figure 14 ). Inshore transplanted corals experiencing a greater rate of SWT increase as well as higher SWTs would therefore be expected to upregulate eIF3H to counteract ther mal stress. However, upregulation was only observed in corals at the offshore site. Hence, we observed significant differences in the expression of eIF3H between sites, but our experimental design did not reveal any temperature related stresses in 2012. S imilarly, temperature dependent effects on eIF3h were not evident in the summer of 2013. Based upon the similar SWTs and SWT variation at both sites prior to sampling , we anticipated corals from both sites to display similar expressions or that corals tra nsplanted to the inshore site would be less impacted due to the larger variation in annual temperatures that these corals are accustomed to. Instead, corals from the inshore site displayed greater expression of eIF3H compared to the offshore site contradic ting our expectations. Several factors may have contributed to the unexpected expression patterns of eIF3H. First, the temperature stress experienced during and prior to sampling may not have been significant enough to elicit a response driven by tempe rature. Temperatures prior to sampling did not reach the maximum 67 observed duri ng either 2012 or 2013 (Figure 14 ). Second, eIF3H is likely to be activated by other stressors (e.g. irradiance) in addition to temperature. Therefore insightful applications of this gene may be more appropriate in laboratory settings, rather than in field whe re environmental conditions can not be controlled. 2.5. 3 Adaptive respons e to immune system activation: adenylate cyclase associated protein 2 (ACAP2) Through interactions with activated Ras proteins, adenylate cyclase associated proteins (ACAPs) regulate the synthesis of cyclic adenosine monophosphate (cAMP) by adenylate cyclase (AC) (Shima et al., 2000) . When synthesized after Ras coupled activation (Gibbs and Marshall, 1989) , cAMP acts as a potent regulator of the inflammatory response (Serezani et al., 2008) . Therefore ACAP2 reduces over - activation of the innate immune system. Porites astreoides colonies originating from the offshore site expressed significantly greater levels of ACAP2 in all sampling periods relative to those from the inshore site. This result was observed independent of transplant location, indicating that corals inhabiting the offshore site may b e locally adapted to this environment. Local adaptation requires very limited gene flow between populations along with intense selection on the variation of a phenotype (Kawecki and Ebert, 2004) . The broad dispersive reproduction strategy used by corals in addition to the long lived nature of reef building corals has long been thought of as a barrier to local adaptation in the host animal. Wide dispersal facilita tes consistent low - level gene flow between metapopulations and serves to increase diversity (Sammarco and Andrews, 1989, 1988) . Although phenotypic diversity is necessary to produce local adaptation, di versity can also decrease the effect of selection pressure on a particular phenotype (Sanford and Kelly, 2011) . For instance, in our s tudy significant site of origin - dependent dif ferences in 68 ACAP2 expression suggest local adaptation. Supporting our assumptions, Kenkel et al. (2015) observed very little gene flow between inshore and offshore populations of P. astreoides inhabiting the FRT. Local adaptations confer fitness advan tages to an organism that is confronted with a hostile environment. We have previously established, based on expression of TRAF3 , that the offshore T herefore our results indicate that the offshore environment exposes corals to increased biotic stress. Acute and chronic inflammatory responses act to the detriment of the host by decreasing fitness. For example, inoculation of the mealworm beetle ( Teneb rio molitor ) with non - lethal levels of bacteria has been shown to decrease longevity (Moret and Siva - Jothy, 2003) . Moreover, individual s of T. molitor that produce elevated levels of melanin, a critical molecule in the invertebrate innate immune response, have decreased longevity even without external stimulation (Armitage et al., 2003) . Thus reducing overactive or continuously activated immune responses may confer an advantage to the host. TRAF3 expression (i.e. an inflammatory response) was greatest among coral fragments transplanted offshore, which provides compelling evidence for an adaptive respons e to immune system stimulation. Organisms m ust allocate resources to all cellular processes from a finite supply, however, immune responses come with a resource cost (Lochmiller and Deerenberg, 2000) . It is therefore common to observe tradeoffs (i.e. costs), in which the expressio n of an adaptive trait increases while the expression of another trait or traits decreases. Tradeoffs resulting from adaptive responses to the innate immune system are more commonly detected in higher level traits such as survival, growth, longevity, and fecundity (Lochmiller and Deerenberg, 2000; Sheldon and Verhulst, 1996) because of the difficulties inherent to observing the intera ctions between lower level traits. Coral species inhabiting the offshore reefs of the FRT, including P. astreoides , display both a decreased mean colony size as well as decreased abundance relative to the populations inhabiting inshore reefs (Haslun et al ., 2015). Moreover, colony 69 size is correlated with increased fecundity in P. astreoides (Chornesky and Peters, 1987) . Porites astreoides colonies originating from the offshore sites in our study averaged 7.34 cm in diameter while those inhabiting the inshore site were 11.44 cm i n diameter (Haslun et al., 2015). A decrease in the rate of skeletal linear extension (i.e. growth) and decreased fecundity are likely to be a high level fitness tradeoff associated with diverting resources to both an active immune response and counteract ion of that immune response. Similarly, P. astreoides colonies inhabiting an adjacent offshore site have been shown to display decreased growth following exposure to stress relative to corals collected from an inshore reef (Kenkel et al., 2013a) . My r esults indicate that the activation of the immun e response has affected population dynamics of P. astreoides . T he response of the host to activate the immune system at offshore sites likely resulted in an opposing adaptive response and decreased host growth rate and fecundity . To reiterate, I observed decreased colony sizes at the offshore site that is consistent with this observation. Although there is no doubt that climate related changes in abiotic factors (e.g. SWT) act to the detriment of corals and may also contribute to local adaptatio n, the interaction of abiotic and biotic stressors is important and should response, be it innate or other, may be especially important along reefs tha t have been drastically impacted and continue to be impacted by disease, such as the FRT (Porter et al., 2001 ) . 2.6 C o n c l u s i o n My st udy shows that activation of the immune system in P. astreoides differs between an adjacent inshore and offshore reef. Colonies that were transplanted offshore expressed TRAF3 more than colonies that were transplanted inshore. Activation of TRAF3 occurs following recognition of foreign substances by TLRs, and therefore increased expression is likely a result of increased stress brought on 70 from biotic sources (i.e. bacteria and viruses). P. astreoides originating from the offsho re environment also displayed increased expression of ACAP2 independent of any transplantation, an indication of local adaptation to stress. Because increased expression of the immune system results in fitness tradeoffs, this particular adaptation may be an effort by corals to enhance survival by depressing activation of the immune response brought on by the offshore environment. P. astreoides inhabiting the offshore reef not only activated their immune pathways but also regulated this response by express ing ACAP2. While this likely enhances survival, it limits resource availability for other traits such as growth. Offshore bank reefs throughout the FRT currently exhibit decreased mean colony size and decreased abundance relative to the inshore and our s tudy provides evidence indicative of a link between increased immune responses and fitness. Increased immune system activation was associated with decreased SWT variation. Offshore sites displayed a milder temperature regime compared to inshore sites. Because lowest TRAF3 expression was observed during a period of decreasing winter SWTs, lower winter temperatures may decrease the activation of the immune system by limiting sources of biotic stress. As climate warming continues, winter low temperatures will likely increase placing biotic stress on inshore reefs and further exacerbate warming at offshore reefs. The resulting increase in immune system activation may place resource more severe bleaching events than currently occurs. 71 CHAPTER 3 DIVERGENT RESPONSES OF PORITES ASTREOIDES POPULATIONS TO BACTE RIAL ENDOT OXIN: POTENTIAL CONSEQUENC ES OF IMMUNE SYSTEM ACTIVA TION 3.1 A bstract Diseases have greatly impacted coral reefs of the Florida Reef Tract (FRT) causing changes to reef community structure. These changes have been realized in the form of mass mortalities of particular species. While the direct effects of disease are import ant, indirect effects of bacterial related stress have received less attention in the literature despite documented links between immune system activation and fitness related traits in invertebrates. Currently the inshore patch reef zone of the FRT contai ns more coral cover and larger coral colonies than the offshore bank reef zone despite increased exposure to thermal stress at the inshore site. The response of Porites astreoides originated from an inshore reef (n = 6) and offshore reef (n = 6) to a cont rol condition (28°C), elevated seawater temperature (SWT; 32°C), and the synergistic effects of elevated SWT and bacterial lipopolysaccharide ( 5 µg mL - 1 ) was evaluated. The expression of two genes affected by SWT ( eukaryotic translation initiation factor 3, subunit H ; heat shock factor protein 1) and two genes affected by pathogens ( TNF receptor associated factor 3 ; calcium dependent protein kinase) was quantified with quantitative real - time reverse transcriptase poly merase chain reaction (qRT - PCR). Two g ene regulation strategies were identified, which reflected site of origin - dependent variation in either SWT or biotic stress . Offshore corals displa yed increased expression across all 4 genes to elevated SWT and LPS but not elevated SWT alone compared to the control treatment. I nshore corals displayed increased expression to elevated SWT but not to the synergistic treatment, compared to the control . Offshore fragments of P. astreoides also displayed increa sed gene expression compared to inshore fragments at the control treatment . While these results indicate that P. astreoides may be a highly adaptable coral species, they also indicate the potential for adaption to a single stressor. Increased response to bacterial stress was associated 72 with decreased c oral abundance and size indicating potential tradeoffs that constrain fitness related traits. The importance of synergism between sea water temperature and microbial related stress deserves increased attention as a driver of coral community dynamics. 73 3.2 I ntroduction Diseases of terrestrial and aquatic species can drastically affect ecosystem function as a consequence of mass mortality, decreased size distribution, and range shifts (Crowl et al ., 2008) . Marine pathogens are recognized for their ability to restructure communities across habitats (Burge et al., 2014) . Examples include Dermo and Multinucleate Sphere X diseases of oyster populations along the east coast of the United States (Powell et al., 2008) , an unidentified disease of the spiny sea urchin Diadema antillarum in the Caribbean (Lessios et al., 1984) , white pox of Acropor a spp. coral throughout the Caribbean (Gladfelter, 1982) . Nov el diseases continue to emerge (Hewson et al., 2014) . The importance of interactions between the impacts of marine epizootics and climate is becoming increasingly recognized. Increases in the a bundance of pathogenic planktonic bacteria (e.g. Vibrio spp. ) in response to increased sea surface temperature has occurred in the southern North Sea over the past 50 years (Vezzulli et al., 2 012) . As climate shifts, diseases affecting reef building corals are also likely to increase and cause as much mortality as temperature induced bleaching (loss of symbiotic algae) in the next 20 years (Maynard et al., 2015) . The primary causes of this outcome are changes in host susceptibility and behavior as well as changes in pathogen virulence and grow th (Maynard et al., 2015) . Caribbean reefs display the greatest frequency of infection and diversity of d iseases relative to reefs worldwide (Garzón - Ferreira et al., 2001; Weil and Rogers, 2011) . Many diseases are uncharacterized. Such diseases do not have an etiological agent but are important infectious agents in the Caribbean (Weil and Rogers, 2011) . The a croporiid corals, once the dominant coral cover in the region, are now functionally extinct along the reef trac t as a result of the enteric pathogen Serratia marcescens (Gladfelter, 1982) . The prolific grazing sea urchin, Diadema antillarum , was similarly extirpated from this region by an unknown 74 pathogen (Lessios et al., 1984) . This result ed in increased algal growth that further decreased coral cover. At present approximately 70 % of coral cover in the Florida Keys consists of inshore patch reefs rather than the offshore bank reefs that dominated historically. Additionally, offshore bank reef coral communities display decreased colony size and decreased species richness, indicative of a more detrimental environment than currently found inshore (Lirman and Fong, 2007) . Site - dependent differences in the response of the host to detrimental levels of abiotic stress do not appear to correlate well with patterns of growth (Manzello et al., 2015) and community structure (Haslun et al., 2015) . Because disease prolifera tion is dependent upon host - pathogen interactions, both host and pathogen characteristics may be affected by the environment changing those interactions (Burge et al., 2014) . In marine habitats , temperature (Maynard et al., 2015; Ward et al., 2007) and nutrient regime (Vega Thurber et al., 2014) are hypothesized to be the primary agents of reef decline. However increased s e awater temperature and elevated nutrient concentrations are more strongly evident in inshore patch reefs (Lirman and Fong, 2007) . Inshore sites typically experienc e a 1°C increase in mean maximum seawater temperature (SWT) during summer months and a decrease in mean minimum SWT by 1°C as well as increased turbidity and nutrients relative to the offshore . Instead, the synergistic effects of temperature stress and overexpression of defenses against microbial pathogens may be significant factors detrimentally affecting offshore corals to a greater extent than the inshore populations. Therefore including bioti c stressors in the estimation of host responses to environmental change provides a better representation of stresses driving declines in coral cover, diversity, and colony size, than thermal stress alone. The innate immune system is the primary defense of the coral metazoan host against biotic stressors. Continued activation of the innate immune system can, however, result in a compromised state that can exacerbate the effects of previously benign conditions due to resource over - allocation. In this stu dy I 75 e xplore the e ffect of environmental history on the response of Porites astreoides to thermal and bacterial stress. A lipopolysaccharide (LPS) endotoxin from Serratia marcescens , a known coral pathogen, was applied to induce a response in dicative of ba cterial stress. We hypothesize that corals inhabiting offshore sites display a greater stress response to LPS than inshore communities. As previously noted, disease attributed to S. marcescens infection has impacted the offshore bank reef system more tha n the inshore patch reef system (Patterson et al., 2002) . Therefore, we predict that previous or continued exposure to pathogens will result in greater upregulation of the stress response. Corals inhabiting offshore bank reefs are expected to display greater u pregulation of genes compared to conspecifics inhabiting inshore habitats. Our results support this hypothesis and moreover indicate divergent responses of inshore and offshore populations of Porites astreoides to bacterial endotoxin and temperature. 76 3 . 3 M ethods 3.3.1 Colony collection and m aintenan ce The two sites selected for this study are in the lower region of the Florida Keys National Marine Sanctuary : Birthday r eef ( - ) and Acer 24 r eef ( - W ) (Figure 1 9 ). Birthday reef is representative of inshore patch reefs while Acer 24 reef is representative of offshore bank reef environments. Both sites contain populations of P. astreoides that were sampled with minimal impact to the population (sampl ing affecting < 1 % of the population) ( Erich Bartels pers. comm.) . T ransplant experiments at each site were conducted at a depth of approximately 6 m. Therefore, differences in light level as a function of depth was not a factor affecting the responses of coral colonies. Differences in coral cover and colony size between Acer24 and Birthday reef has been previously reported (Haslun et al. , 2015 in review ) and follow a trend of increased coral cover and colony size with decreasing distance from shore. 77 Figure 1 9 : The Inshore Patch R eef (Birthday Reef; - ) and Offshore Bank R eef (Acer 24 Reef; - W ) Sampling S ites are Pictured A bove 78 Fragments of Porites astreoides colonies (n = 6), 16 x 16 cm, were collected with a cold chisel and mallet by divers using SCUBA (NOAA National Marine Sanctuaries Permit # FKNMS - 2011 - 10). F ragments were transferred to Mote Marine Laboratories Tropical Research Laboratory in coolers f il led with site derived water and upon arrival sectioned in half to provide samples for a companion study. The fragment halves were allowed to equilibrate for three days in raceways supplied with a constant flow of oceanic seawater and protected by shade clo th. Sampled were secured to concrete:aragonite (1:3) disks (radius 6 cm) with All Fix Epoxy (Cir - Cut, Lafayette Hill, PA) and equilibrated for two days in the raceway prior to being transplanted in the field. Porites fragments were affixed to submerged c oncrete cinder blocks with All Fix Epoxy (NOAA National Marine Sanc tuaries Permit # FKNMS - 2011 - 10) at their site of origin or at the companion site, either Birthday reef or Acer 24. The random effects associated with the spatial distribution of sampled co lonies within the benthic habitat were reduced by collection and subsequent transplantation back to the site of origin on a clean concrete substrate. Settlement position can impact the abiotic and biotic regime experienced by corals due to habitat heterog eneity, light levels, hydrodynamic differences, and interspecific interactions. Transplanting coral fragments back to the site of origin upon a known substrate at the exact same depth served to decrease potential differences associated with settlement dep endent factors. Further, by allowing these coral fragments to remain at the site of origin for two years all fragments experienced very similar environmental conditions at a given site. Further details regarding sampling and transplantation can be retrie ved from a previous manuscript (Haslun et al., 2015 in review) . After the two - year period of transplantation each colony was collected and returned to the Mote Marine Laboratory Tropical Research Station and returned to a raceway. Colonies were then al lowed to recover from collection for two days at which point each fragment was cut with brick saw into nine 2.54 79 x 2.54 cm fragments. Sterile seawater was used as a lubricant to decrease heat associated with the cutting wheel. Each set of fragments from was allowed to recover for at least 3 days in the seawater raceway prior to experimentation. 3.3 .2 Laboratory e xperiment Two treatments and a control were used to evaluate the effect of collection site on the response of Porites astreoides to temperature and a bacterial endotoxin. The control environment of 28°C was chosen because it reflects the temperature during the transition from summer to winter when temperatures at these two sites is equivalent. The first of the two treatments simulated a temperat ure of 32°C. During the two - year field transplantation period this was the highest one - day mean temperature observed. Therefore we expected that it would reflect a realistic but stressful condition. The second treatment was chosen to evaluate the synerg istic effect of high temperature (32°C) and exposure to a bacterial endotoxin. Lipopolysaccharide from Serratia marcescens ATCC 21639 (Sigma - Aldrich; St. Louis, MO) was applied at a final concentration of 5 µg mL - 1 ; a level previously chosen to evaluate endotoxin impacts on corals (Palmer et al., 2011) . A bacterial endotoxin treatment at 28°C was not conducted fo r three reasons. First and foremost, sectioning each coral fragment into 9 samples limited the number of treatments possible while preserving an adequate sample size at the individual level (n = 3). Second, minimizing the environmental impact of addition al coral collection was a concern in this region. Lastly, the aim of this study was to understand site dependent responses to the synergistic effects of temperature and LPS stress rather than the response to LPS across a range of temperature stress. Exp eriments were carried out in acrylic boxes (5 x 5 x 18 cm) constructed from 0.030 cm thick acrylic sheet. Acrylic boxes contained 250 mL of sterile artificial seawater and each box separated one coral 80 fragment from another assuring an independent response of each fragment to the experimental treatment. Groups of fifteen boxes were placed in 37.85 L glass aquariums filled with sterile seawater, which served as temperature incubation chambers. Control and treatment temperatures were maintained with a 150 W adjustable aquarium heater (Eheim; Dollard - Des - Ormeaux, Quebec ) . A submersible Aquaclear 10 aquarium powerhead (Hagen; Mansfield, MA) served to circulate water and maintain a homogenous thermal profile. Each aquarium containing 15 acrylic boxes was covered by shade cloth throughout the course of an experiment resulting in a maximum photosyntheti cally active radiation of 150 µ mol photons m - 2 s - 1 . This level was similar to that experienced at each reef and is a level known to prevent photoinhibition of reaction centers in Symbiodinium spp. dinoflaggelate symbionts. Each experiment lasted for 8 h beginning at 9:00 AM and concluding at 5:00 PM the following day. Colony fragments in acrylic boxes were randomly distributed within an aquarium for a given t reatment (n = 3) to avoid bias associated with variation of conditions within an aquarium. Following 8 h treatment, each section of a fragment w as immediately frozen in liquid nitrogen (L N 2 ) and stored at - 80°C until processing for quantitative reverse tr anscription PCR. 3.3 .3 Sample processing C oral fragments fixed with L N 2 were first pulverized into smaller sections using a hardened steel dounce - style homogenizer chilled with liquid nitrogen to retain sample integrity. Excess skeletal fragments were rem oved with forceps and then the remaining portion ground using a ceramic mortar and pestle. In order to prevent RNA degradation during processing, fragments free of extraneous debris, were crus hed in a shallow pool of L N 2 . The powder was then transferred to a microcentrifuge tube and stored at - 80°C prior to RNA isolation. Mortar and pestles were cleaned with Alconox (Alconox; White Plains, NY), 81 rinsed three times with de - ionized water, RNase treated (RNase zap: Sigma - Al drich, St. Louis, M O), and then rinsed with ultra - pure deionized water (E - Pure System; Thermo Fisher Scientific Inc.) before each subsequent sample was processed. 3.3 .4 RNA i solation RNA was isolated from sample powder (110 mg) using a mixture of guanid ine thiocyanate and phenol in a monophase solution (TRI Reagent: Sigma - Aldr ich, St. Louis, MO). TRI Reagent (1 mL) was added to the sample and aspirated to aid in cell disruption prior to incubation (10 min, room temperature). The sample was centrifuged ( 10,000 rcf, 10 min, 4°C ). The supernatant was transferred to a new tube and a solution of 0.8 M sodium citrate: 1.2 M sodium chloride added ( 250 µL ). The sample was s haken vigorously for 5 sec . I sopropanol (neat, 250 µL ) was added and tube shaken vigorous ly for 15 sec . Samples were incubated (10 min, room temperature) to precipitate RNA and then centrifuged (10,000 rcf, 10 min, 4°C ) . After decanting the supernatant , the RNA pellet was washed with ethanol (1 mL, 75%) and centrifuged (7,500 rcf, 5 min , 4°C ) . This step was carried out twice. The extracted RNA was dissolved in RNase and DNase - free water ( 100 µL ) and integrity determined with a Caliper Lab Chip GX. RNA quality scores (RQS) greater than 6 were deemed of sufficient quality for two - step reverse transcription quantitative real - time polymerase chain reaction (qRT - PCR) (Fleige an d Pfaffl 2006) . RNA extractions yielded approximately 2 µg of RNA ( 110 mg sample - 1 ) . 3.3 .5 Two - s tep qRT - PCR Isolated RNA (300 ng) was treated with 1 unit of DNase 1 (Life Technologies; Grand Island, NY) in endations. The sample ( 260 ng ) was reverse transcribed with the Superscript III first strand synthesis supermix (Life T echnologies; Grand Island, NY) in 96 - well plate s on an Eppendorf Mastercycler (Eppendorf; Hauppauge, N Y). The thermal profile was as fo llows: 82 10 min,25°C; 30 min,50°C; 5 min, 85°C. After denatu ring the enzyme at 85°C, RNase H was added (1 µL) to each well and incubated (37°C, 5 min ) to degrade the remaining template RNA. To each cDNA product (20 µL), 7.5 M ammonium acetate (Sigma Aldr ich; St. Louis, MO) was added (6 µL) followed - 20°C isopropanol (neat, 50 µL) . This solution was inverted 10 times to mix t he contents and then chilled ( - 80°C, 1 h ) to precipitate cDNA. Samples were centrifuged (18,000 rcf, 15 min , room temperature) to pellet the cDNA. The precipitation reagents were decanted and p ellet washed twice with 75% ethanol (1 mL) . Washed cDNA was centrifuged ( 18,000 rcf , 10 min, room temperature) . After the second wash the pellet was allowed to air dry at room temperatur e for 10 - 15 min. The pellet was re - suspended in ultra - pure water (104 µL) to yield a final cDNA concentration of 2.5 ng µL - 1 . This cDNA was stored at - 20°C for no more than 1 week prior to qRT - PCR. 3.3 .6 qRT - PCR primer v alidation Sequences of transcript s of interest were obtained from the Porites astreoides SymBioSys database ( http://sequoia.ucmerced.edu/SymBioSys/ ). Primers were created with Primer3 software applying selection criteria previously o utlined (Haslun et al. 2015 in review ). The top scoring primer pair was selected for qRT - PCR validation. Primer validation was performed by creating a standard curve relating the cycle of quantitation (Cq) to the log concentration of cDNA along a 2 - fold s erial dilution; 5 ng µL - 1 , 2.5 ng µL - 1 , 1.25 ng µL - 1 , 0.625 ng µL - 1 , and 0.3715 ng µL - 1 of cDNA. Primer pairs amplifying a single product according to dissociation curves, displaying a highly linear relationship (R 2 > 0.99) and adequate amplification efficiency (3.0 < E < 3.6) were consid ered valid for analysis (Table 6 ). The genes of interest (GOI) included 2 genes previously utilized in a sister study (Haslun et al. 2015); Eukaryotic initiation factor 3 subunit H (e IF3H), TNF receptor - associated fac tor 3 (TRAF3). Two additional genes of interest were also included; Calcium calmodulin dependent protein kinase (CDPK) and Heat shock factor protein 83 1 (HSFP1). We included two housekeeping genes, 60S ribosomal protein L11 (RPL11), and Cathepsin L (CATL) a s utilized in previous work on gene expression with this species ( Kenkel et al., 2011 ; Haslun et al. 2015 in review ). Table 6 : The amplification efficiency and primer sequences of each gene of interest and control gene investigated in this study. Genes of Interest Abbreviation Primer Sequ - Efficiency Calcium calmodulin dependent protein kinase CDPK F: TCAAGCATAAGTGGGTGCAG R: ATAGCCAACATTCCGCCTTT 1.91 Eukaryotic initiation factor 3, subunit H EIF3H F: TTGATTGATACCAGCCCACA R: ACAAACTGCTTTGCTTTCCC 1.97 Heat shock factor protein 1 HSFP1 F: CTGCTTTGCCAGATGATGAC R: GGGCTGTGATGTTGAAGGA 1.95 TNF receptor - associated factor 3 TRAF3 F: GTCTGGCTCCTCCCATCTTT R: GCCTCCAGCATTCTAACCTG 2.03 Control Genes 60S ribosomal protein L11 RPL11 F: TTTCAAGCCCTTCTCCAAGA R: GACCCGTGCTGCTAAAGTTC 1.94 Cathepsin L CATL F: GGAAGGATTACTGGCTGGTC R: GGATAGATGGCGTTTGTGG 2 Real - time PCR reactions were c onducted in 10 µL total volume (1 µL template cDNA, 5 µL Power SYBR green master mix (Life Technologies ; Grand Island, NY), 1.5 µL primer pair (final concentration 250 nM), and 2.5 µL DNase and RNase - free water ) . Amplification and detection of transcripts was carried out on an Applied Biosystems 7900HT real - time PCR system in 384 - well plate format following the mmendations. To prevent potential run to run variation and the need for inter - run calibration, all samples were analyzed in duplicate on a single 384 - well plate for each gene of interest (Derveaux et al., 2010) . 84 3.3 .7 qRT - PCR a nalysis A Bayesian model - ba sed approach (Matz et al., 2013) was applied to Cq values based upon previous gene expression studies with Porites astreoides (Matz et al., 2013) . This method converts Cq values to molecular counts creating a linear rather than exponential relationship for the response variable. We employe d a 2 - way factorial design including collection site (inshore patch reef and offshore bank reef) and treatment as factors. Equation 1 outlines the structure of the model in the MCMCglmm package (Matz, 2013) gene Col Treatment represents the level of expression associated with a given factor level ( i.e. control, temperature, temperature + biotic stress). Conditions in brackets indicate random err or terms and include the sample specific error, the gene specific sample error, and the gene specific error in the order presented in each model. Equation 1: Much like traditional delta delta Cq methodology (Livak and Schmittgen, 2001) we chose to include reference genes in this model to control for variation associated with the e xperimental conditions. This adjustment is not necessary in a Bayesian model - based approach, but provides more precise estimates of transcript abundance across treatments. 85 3.4 R esults 3 .4.1 Control c ondition (28°C) Porites astreoides sections experiencing the control environment (28°C) displayed the lowest transcript abundance across all genes of interest (GOI) at each site. Comparison of transcript abundance between sites i ndicated that express ion significantly differed for e IF3H (F igure 20) , HSFP1 (Figure 21) , and TRAF3 (Figure 22) (p < 0.0). Mean gene expression in fragments originating from the offshore site (Acer 24) was 3 times greater than that of conspecifics from the inshore site ( Birthday Reef ) . Although control levels of CDPK (Figure 23) did not signif icantly differ between sites the means followed this trend. Figure 20 : e IF3H Transcript Abundances A ssociated W ith Porites astreoides Fragment Origination at an Inshore or Offshor e Reef Site Following 8 h Incubation With One of Three Treatments; 28°C (C ontrol) (28) , 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens . Points indicate 86 Figure 2 0 posterior means and error bars indicate 95% credible intervals obtained from Bayes ian generalized linear mixed modeling. Figure 21 : HSFP1 Transcript Abundances Associated W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef Site Following 8 h I ncubation With One of Three Treatments; 28°C (C ontrol) (28) , 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens . Points indicate posterior means and error bars indicate 95% credible intervals obtained from Bayesian generalized linear mixed modeling. 87 Figure 22 : TRAF3 Transcript Abundances Associa ted W ith Porites astreoides Fragment Origination at an Inshore or Offshore Reef S ite Following 8 h Incubation W ith One of Three Treatments; 28°C (C ontrol) (28), 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens . Points indicate posterior means and error bars indicate 95% credible intervals obtained from Bayesian generalized linear mixed modeling. 88 Figure 2 3 : CDPK Transcript Abundances Associated W ith Porites astreoides Fragment Origination at an Inshore or Of fshore Reef Sit e Following 8 h Incubation With One of Three Treatments; 28°C (C ontrol) (28), 32°C (32) , and 32°C + L ipopolysaccharide (32L) of Serratia marsescens . Points indicate posterior means and error bars indicate 95% credible intervals obtained fro m Bayesian generalized linear mixed modeling. 3.4.2 Increased t emperature t reatment (32°C) Inshore and offshore fr agments incubated at 32°C for 8 h did not display a significant difference in transcript abundance across the 4 genes of interest. When compared to the control treatment, f ragments collected from the inshore reef displayed a significant upregulation of all genes excep t for CDPK. HSFP1 was upregulate d 3 times that of the control, e IF3H 4 times that of the control , and TRAF3 5 times that of the control. Offshore fragments did not display significant changes in expression compared to the control treatment . 89 3. 4.3 Synergistic effect of temperature and l ipopolysaccharide (32°C + 5 µg mL - 1 LPS) Porites astreoides fragments from the offshore reef displayed greater transcript abundance for all GOIs then inshore conspecifics that w ere exposed to the synergistic e ffects of increased temperature and LPS stress. This difference was 1.75 times greater for CDPK e xpression, 4 times grea ter for e IF3H expression, 3 times greater for HSFP1 expression, and 3 times greater for TRAF3 expression. Exposing offshore fragments to the synergistic stress of increased temperature and LPS resulted in the upregulation of all GOIs relative to both th e control and elevated temperature treatment. Conversely, inshore fragments downregulated gene expression when confronted with the synergistic stress of elevated temperature and exposure to LPS relative to elevated temperature alone. Gene expression of t he inshore fragments was comparable to the basal levels associated with the control condition. 90 3 . 5 D iscussion Previous observations of Porites astreoides populations inhabiting an offshore bank reef indicate a smaller mean colony size, decreased abundance, and increased constitutive expression of adenylate cyclase associated protein 2 (ACAP2), a gene associated with the innate immune response, compared to conspecifics inhabiting the inshore patch reef environment (Haslun et al., 2015) . Because thermal stress from decreased variation in SWTs at offshore bank reefs is unlikely to play a role in the ada ptation of gene expression (Haslun et al., 2015 in review ) in this region I h ypothesized that biotic stress plays a more prominent role in shaping the observed differences in phenotypes and coral communities. We established that P. astreoides colonies inhabiting an inshore and offshore reef of the lower FRT dis play divergent responses to brief thermal stress in a laboratory setting (8 h). We also determined that these two populations of P. astreoides display a comparably greater divergence in their response to the combination of thermal stress and biotic stress from bacterial lipopolysaccharide of a known coral pathogen ( Serratia marcescens ). These results indicate that non - lethal biotic stress may be underrepresented in the literature as a stressor capable of structuring communities especially when the effects of climate change simultaneously considered. Eukaryotic and prokaryotic organisms affect changes in gene expression constitutively or responsively to environmental changes (Kussell and Leibler, 2005) . Constitutive changes in expression are maintained over long periods of time (adaptation) while responsive changes are transient and dependent upon the occurrence of a particular stressor (Geisel, 2011) . The organisms that upregulate a particular gene or suite of genes tend to experience increased fitness (e.g. growth and reproduction) compared to those that do not change regulatio n within a given environment. We observed that coral fragments collected from the offshore habitat displayed a 2 - fold increase in expression of all GOI compared to conspecifics collected from the inshore habitat at the control condition (28°C). At increa sed temperature (32°C) P. 91 astreoides fragments displayed similar levels of expression independent of the collection site. However, at an elevated temperature, P. astreoides collected from the inshore site significantly upregulated gene expression relative to the control treatment across all GOI while fragments collected from the offshore site maintained their level of gene expression relative to the control. This result suggests that corals inhabiting offshore sites constitutively upregulate genes during short - term exposure to thermal stress while corals inhabiting inshore sites employ a responsive gene regulatory strategy. The observed difference in response strategy to temperature may reflect differences in the variability in SWT associated with each si te. As stated previously, inshore patch reefs are exposed to a wider variation in SWT and therefore responsive regulation of gene expression may be an important component to fitness. In yeast, however, increased levels of gene expression are associated w ith significant energy costs (Wagner, 2007, 2005) . A responsive gene expression strategy by P. astreoides to the wide variation in thermal stress, characteristic of inshore patch reefs, may decrease the impact of energy costs on fitness due to constant gene regulation. Thermal preconditioning experiments with corals support this view. Laboratory preconditioning with mild thermal stress has been observed to result in acquired resistance to severe thermal stress in the corals Acropora millepora (Bellantuono et al., 2012) and Acropora aspera (Middlebrook and Hoegh - Guldberg, 2008) . Further, our previous research has shown that the coral Montastraea cavernosa displays an increased susceptibility to thermal stress in environments with lowered exposure to non - lethal thermal stress (Haslun et al., 2011) . Responsive expression to temperature is also common in other benthic metazoan marine species. Following a prolonged period of e xposure to elevated temperatures abalone (Li et al., 2012) and the purple sea urch in Stronglyocentrotus purpuratus (Osovitz and Hofmann , 2005) display responsive increases in the expression of hsp70, a chaperonin protein that decreases the effects of thermal stress. 92 Recent evidence from common garden experiments with juvenile P. astreoides from inshore and offshore reefs shows that th e inshore population displays increased growth rates under thermal stress compared to offshore corals (Kenkel, Setta, and Matz, 2015) . Although the authors conclude that a proportion the observed variation in fitness is attributable to maternal effects, t he species appears to maintain population level genetic variation that provides the necessary material for adaptation to environmental variation. Although it is early to conclude that these adaptations are definitively heritable, our previous research rec iprocally transplanting P. astreoides between the inshore and offshore reefs provides evidence of an occurrence of local adaptation in the expression of ACAP2 (Haslun et al., 2015 in review) . The present study begins to elucidate the relative contribution s of two environmental factors, temperature and LPS, on the phenotypic value (P) and variation (V E ) in four GOI of P. astreoides . The phenotypic value, in this case the mean gene expression, is defined as the sum of genotype driven and environmentally dri ven expression of the observed phenotype. We did not determine the genotype of the individuals used in this study and therefore comment on the effect of the environment only. It should be noted however that the variation associated with a given individua ls expression of a gene was included in the model as a random effect. Moreover, as previously indicated (H aslun et al., in review; Kenkel, Setta, and Matz, 2015) these two regions display differences in traits that indicate distinct populations and theref ore genetic differences are likely. Phenotypic variation (V E ) related to the environment is defined as the additive effect of the general environment (V Eg ), gene by environment interaction (V GxE ) and the microenvironment (V Es ) on the total phenotypic vari ation (Equation 1 ) (Byers and State, 2008) . The contribution of the microenvironment was limited by the experimental design and thus assumed to be negligible. Equation 1 : 93 Corals that experienced the offshore site environment displayed minimal differences in phenotypic value as well as phenotypic variation to both thermal treatments. This result suggests that variation in phenotypic value, the mean transcript abundance, is likely dependent upon genetic variation rather than environmental variation because there is limited change across the temperature range. Therefore, future adaptation to thermal stress may be constrained for the host in offshore populations of P. astreoid es. An alternative explanation to the observed gene expression is that limited acclimation to increased thermal stress occurred as a result of the narrower thermal range characteristic of offshore sites. When combined with the short period of thermal str ess used in this study (8 h), the experiment may not have been carried out across a long enough time span to produce an observable response in the expression of the GOI. In many instances corals spanning a reef display an observable response only after a number of degree heating weeks; weeks with prolonged temperature stress above the mean monthly maximum SWT (Gleason and Strong, 1995) . However, decreased acclimation appears to be an unlikely explanation given that offshore corals displayed incre ased levels of expression at 28 °C comp ared to the expression of inshore corals, which experience a wider range of temperatures. Therefore offshore corals maintained an increased level of expression in all GOI at all times. This strategy could limit resources needed to confront other stressor s and c ontribute to decreased fitness. Although our data indicates that temperature has an impact on corals inhabiting these two regions, the emergence of disease as a driver of ecosystem change is closely linked to the effect of warming climates on micro be pathogenicity and host disease resistance (Burge et al., 2014) . Therefore understanding the effect of this synergistic stress is a critical component to understanding future community dynamics. The strength of the link between disease and en environmental change. Homeothermic organisms are less sensitive to environmental change and therefore the disease dynamics are more impacted by the response of the pathogen to environmental change (Harvell et al., 2009) . Conversely, ectotherms are more sensitive to climate warming thus there 94 is a strong link between host resistance to disease and environmental change. Corals are ectotherms that are highly sensitive to m inor changes in SWT and are therefore an example of an animal in which the link between host resistance and warming is particularly important (Hoegh - Guldberg et al., 2007) . Increased SWTs cause dysfunction i n the photosynthetic apparatus of the algal symbiont resulting in the production of free radicals and potential expulsion of the symbionts from host (bleaching) along several different pathways (Lesser, 1997) . As up to 98 % of the carbon required by the host is translocated from the symbiont (Muscatine et al., 1981) , bleaching reduces the resources available to the host to respond to concurrent or later perio ds of stress from a biotic source. The effect of bleaching on disease susceptibility has observed. Following recovery from a severe thermal bleaching event in 2005 the coral population of the US Virgin Islands was decimated by an epizootic of lethal whit e plague disease. Fifty percent of the total coral population was lost (Miller et al., 2009) . The authors concluded that the loss of resources to bleaching resulted in a compromised state to defend against such a disease. This example demonstrates how an abiotic stressor can de pathogen exposure despite an apparent healthy state. To my observed. Invertebrates rely upon the highly conserved innate immune system to defend against foreign microorganisms. The response to foreign microorganisms is rapid and although invertebrates lack a specific response to a particular microorganism or antigen (adaptive immunity) a non - specific form of memory i s recognized, called trained immunity (Netea et al., 2011) . Trained immunity is defined and a different one (cross - (Netea et al., 2011) . Empirical evidence of trained immunity has been reported across invertebrate phyla including meal worm beetles (Moret and Siva - Jothy, 2 003) , Drosophila melanogaster (Pham et al., 2007) , sponges (Hildemann et al., 1980) , and reef building corals 95 (Vollmer and Kline, 2008) . Vollmer and Kline (2008), who identified trained immunity in corals, also identified a genotype effect on disease resistance in Acropora cervicornis. Therefore, not only does trained immunity occur in invertebrat es but there is also selection for more efficient defense strategies with repeated exposure. Similarly, our previous research identified the presence of site - dependent selection pressure on P. astreoides inhabiting offshore reefs that is capable of produc ing local adaptation in the constitutive expression of the ACAP2 gene (Haslun et al., 2015 in review) . This particular protein affects the production of cyclic adenosine monophosphate, an important second messenger effector molecule in apoptotic cascades, and produces the pro - inflammatory molecule NF - kB, both of which are important components of the immune response. Porites astreoides has also been shown to display levels of melanin production significantly greater than other common coral species followin g exposure to LPS (Palmer et al., 2011) , and therefore may be particularly sensitive to biotic stress. Melanin is a conserved pigment that scavenges free radicals through the pro - phenoloxidase pathway preventing oxidative damage in animals (Grimaldi et al., 2012) . In Cnidarians , melanin is expressed by innate immune system specific cells (amoebocytes) following expo sure to pathogens and climate related stress (Couch et al., 2008) . In this study I determined if the response of P. astreoides to LPS was affected by the previous environmental history in an inshore patch reef and offshore bank reef environment of the FRT. We combined this immune stimulator wi th increased thermal stress (32 °C) because of the documented link between increased disease development and expansion across coral colonies (Cervino et al., 2004 ) and across reefs (Ben - Haim and Rosenberg, 2002) du ring warming periods. We observed divergent responses in P. astreoides fragments to this combined stress dependent upon their site of collection. The phenotypic value of corals collected from the offshore reef was greater following the 8 h treatment than corals collected from the inshore patch reef, which displayed phenotypic values similar to those observed in the control treatment. The site - dependent effect observed here indicates that P. astreoides fragments that had experienced the offshore 96 environme nt displayed a responsive strategy to the combined stressor while fragments inhabiting the inshore site displayed a limited response. These strategies likely reflect a fragments previous exposure to similar stressors. Our previous work identified that P. astreoides inhabiting the offshore site insult, while those inhabiting the inshore site displayed a consistent level of expression (Haslun et al., 2015 in review) . Therefore we infer that the factors contributing to the immune response of the offshore corals are more variable. Increased environmental variation leads to a responsive expression strategy, as was observed in the high temperature treatment for corals inhabiting the inshore site. This result may also indicate a limited potential for the coral host to adapt to bacterial related stress at inshore sites. In order to summarize the response strategies of the two P. astreoides populations across all treatment levels we applied an additive linear model to the phenotype values (mean gene expression). Two different adaptive response strategies to bacterial LPS and thermal stress were evident for P. astreoides (Figure 4). Colonie s inhabiting the inshore site (1) display decreased expression levels in the GOI studied and employ a responsive strategy to thermal stress but a constitutive strategy to LPS and thermal stress. In contrast, (2) colonies inhabiting the offshore site displa y increased expression to all treatments and display a responsive strategy to LPS exposure. The response planes produced in figure 2 4 include an estimate for the expression to LPS exposure at 28°C in order to produce a complete relationship between the tr eatment factors. We determined the estimate for each population by calculating the average expression between the control treatment and the combined stress treatment. Because inshore corals utilize a responsive gene regulation strategy to temperature but not LPS exposure, the estimate of gene expression was similar to the mean of control and combined stress treatment. Corals collected from the offshore site displayed a responsive strategy to LPS but not to thermal stress, 97 therefore the estimate of gene e xpression when exposed to LPS at 28°C fell between that of the combined stress treatment and control treatment. The proposed relationship, when combined with the known abundance and size distribution of the inshore and offshore reefs, show that responsive expression to thermal stress is correlated to increased growth and abundance while responsive expression to bacterial LPS is correlated with decreased growth and abundance. Therefore responsive confer fitness costs to P. astreoides . The general increased activation of genes may also contribute to decreased phenotypic values of fitness related traits as previously indicated for growth. Figure 24 : The Mean Gene E xpression Response Strategies t o Temperature and L ipopolysaccharide of Porites astreoides Inhabiting Inshore Patch Reefs and Offshore Bank R eefs of the Florida Reef Tract. Each three - dimensional surface represents a 10 x 10 matrix of predicted values from a linear model. The color of each point represents the mean transcript abundance identified in this study. Offshore corals 98 upregulate based on bacterial stress while inshore corals upregulate in response to temperature. Offshore corals also display increased expression compared to inshore corals. Organisms that undergo adaptation to environmental change experience accompanying tradeoffs in the expression of other traits as a result of altered resource allocation. These tradeoffs are more likely observed in higher level traits such as growth, repro duction, and longevity because of the difficult nature of detecting gene regulation interaction. Adaptations to increased biotic stress or an increased range of biotic stress could potentially produce such an effect. Tradeoffs between immune defense and fitness have been observed in other invertebrates. Bacterial challenge of the immune system of Tenebrio molitor, the mealworm beetle, was found to significantly decrease individual longevity (Armitage et al., 2003) . Tenebrio molitor also displays two color morphotypes , tan and black. The 99 darkness of this color is directly related to the production of melanin, an important component of arthropod and coral immune defense. Black morphotypes, which constitutively increase the production of this defense molecule, displaye d decreased longevity (Armitage et al., 2003) . Therefore fitness tradeoffs related to the production of defense molecules can even be produced without microbial attack. Our study indicates that offshore fragments of P. astreoides display both constitutive and responsi ve upregulation of genes following exposure to LPS while when exposed to the same treatment, inshore fragments maintain control and lower levels of expression. Therefore relative to the meal worm beetle, offshore corals display similar expression characte ristics of the immune system and the tradeoffs associated with such a response should also be observed. We have described several sources of evidence corroborating such a fitness tradeoff and therefore biotic stress and its connection with SWT at inshore and offshore reefs requires increased attention. 100 3.6 C o n c l u s i o n This study found evidence indicating that biotic stress is an important driver of host adaptation in and thus community dynamics in the lower region of the FRT. Moreover, previo us studies analyzing fitness of P. astreoides in this region links fitness tradeoffs with increased immune responses that we identify in P. astreoides . Based on our findings we predict that offshore populations of P. astreoides will continue to experience a chronic level of biotic stress that places constraints on their ability to resist other stressors. This population also displays a limited range of gene expression in response to thermal stress indicating constraints on future adaptation to this stress or. With continued climate warming marine microbial communities, particularly thermodependent pathogenic bacteria, are expected to increase in abundance (Bally and Garrabou, 2007) placing greater stress on offshore populations. Although the h, the limited variation in response to LPS that we identified indicates decreased genetic variation for adaptation to biotic stress. If microbial communities continue to transition, inshore microbial communities may generate disease outbreaks that this p opulation is unable to respond to. In the face of continued global warming the importance of investigating climate related e ffects on coastal marine microbial communities cannot be stressed enough. 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