Acute turbidity exposures with Port of Miami sediments impact Orbicella faveolata tissue regeneration.

We evaluated acute turbidity effects on a threatened coral species (Orbicella faveolata) under three short-term challenge scenarios using a Port of Miami sediment homogenate to simulate turbid conditions during dredging. For these experiments we designed a simple coral challenge test system that kept turbidity stable, without adverse effects to the coral. A 96-h coral challenge experiment demonstrated that low turbidity levels (≥4 NTU) have negative effects on O. faveolata tissue regeneration. A 48-h turbidity exposure (maximum 30 NTU) had no effect on O. faveolata tissue regeneration, showing that short term turbidity exposures may not be detrimental to coral health. In a 13-day test, treated coral fragments (maximum 30 NTU) exhibited significant delays in tissue regeneration, but recovery was observed after approximately one week. The results presented here can be used to inform management decisions for proposed dredging activities proximal to coral reef habitats.

Rapid prototyping for quantifying belief weights of competing hypotheses about emergent diseases.

Emerging diseases can have devastating consequences for wildlife and require a rapid response. A critical first step towards developing appropriate management is identifying the etiology of the disease, which can be difficult to determine, particularly early in emergence. Gathering and synthesizing existing information about potential disease causes, by leveraging expert knowledge or relevant existing studies, provides a principled approach to quickly inform decision-making and management efforts. Additionally, updating the current state of knowledge as more information becomes available over time can reduce scientific uncertainty and lead to substantial improvement in the decision-making process and the application of management actions that incorporate and adapt to newly acquired scientific understanding. Here we present a rapid prototyping method for quantifying belief weights for competing hypotheses about the etiology of disease using a combination of formal expert elicitation and Bayesian hierarchical modeling. We illustrate the application of this approach for investigating the etiology of stony coral tissue loss disease (SCTLD) and discuss the opportunities and challenges of this approach for addressing emergent diseases. Lastly, we detail how our work may apply to other pressing management or conservation problems that require quick responses. We found the rapid prototyping methods to be an efficient and rapid means to narrow down the number of potential hypotheses, synthesize current understanding, and help prioritize future studies and experiments. This approach is rapid by providing a snapshot assessment of the current state of knowledge. It can also be updated periodically (e.g., annually) to assess changes in belief weights over time as scientific understanding increases. Synthesis and applications: The rapid prototyping approaches demonstrated here can be used to combine knowledge from multiple experts and/or studies to help with fast decision-making needed for urgent conservation issues including emerging diseases and other management problems that require rapid responses. These approaches can also be used to adjust belief weights over time as studies and expert knowledge accumulate and can be a helpful tool for adapting management decisions.

Assessment of sediment porewater toxicity in Biscayne National Park with sea urchin (Lytechinus variegatus) embryos.

The sea urchin embryo development toxicity test was used to investigate toxicity of the benthic substrate in Biscayne National Park (BISC). Twenty-five sites were selected based upon a high potential for anthropogenic stressor input (e. g., hydrocarbons, personal care products, nutrients, etc.) or proximity to coral reef habitats. We found that sediment interstitial water (porewater) was toxic to urchin embryos at 22 of 25 sites. Healthy sites included two coral reefs (Anniversary Reef and Marker 14 Reef) and Turkey Point Channel. Discrete areas of BISC have highly toxic sediments and the presence of sediment contaminants could negatively impact reproduction, growth and population density of benthic invertebrates, such as corals. Results of the sea urchin embryo development toxicity test can be used as a baseline assessment for monitoring improvements or degradation in ecosystem health and could be a valuable tool to investigate the suitability of degraded habitats for future reef restoration. Since the last comprehensive environmental assessment of BISC was performed in 1999, further investigation into the sources of toxicity at BISC is needed.

Beach showers as sources of contamination for sunscreen pollution in marine protected areas and areas of intensive beach tourism in Hawaii, USA.

In 2019, sands in nearby runoff streams from public beach showers were sampled on three islands in the State of Hawaii and tested for over 18 different petrochemical UV filters. Beach sands that are directly in the plume discharge of beach showers on three of the islands of Hawaii (Maui, Oahu, Hawai'i) were found to be contaminated with a wide array of petrochemical-based UV-filters that are found in sunscreens. Sands from beach showers across all three islands had a mean concentration of 5619 ng/g of oxybenzone with the highest concentration of 34,518 ng/g of oxybenzone at a beach shower in the Waikiki area of Honolulu. Octocrylene was detected at a majority of the beach shower locations, with a mean concentration of 296.3 ng/g across 13 sampling sites with the highest concentration of 1075 ng/g at the beach shower in Waikiki. Avobenzone, octinoxate, 4-methylbenzylidene camphor and benzophenone-2 were detected, as well as breakdown products of oxybenzone, including benzophenone-1, 2,2'-dihydroxy-4-methoxybenzophenone, and 4-hydroxybenzophenone. Dioxybenzone (DHMB) presented the highest concentration in water (75.4 ng/mL), whereas octocrylene was detected in all water samples. Some of these same target analytes were detected in water samples on coral reefs that are adjacent to the beach showers. Risk assessments for both sand and water samples at a majority of the sampling sites had a Risk Quotient > 1, indicating that these chemicals could pose a serious threat to beach zones and coral reef habitats. There are almost a dozen mitigation options that could be employed to quickly reduce contaminant loads associated with discharges from these beach showers, like those currently being employed (post-study sampling and analysis) in the State of Hawaii, including banning the use of sunscreens using petrochemical-based UV filters or educating tourists before they arrive on the beach.

Linking photoacclimation responses and microbiome shifts between depth-segregated sibling species of reef corals.

Metazoans host complex communities of microorganisms that include dinoflagellates, fungi, bacteria, archaea and viruses. Interactions among members of these complex assemblages allow hosts to adjust their physiology and metabolism to cope with environmental variation and occupy different habitats. Here, using reciprocal transplantation across depths, we studied adaptive divergence in the corals Orbicella annularis and O. franksi, two young species with contrasting vertical distribution in the Caribbean. When transplanted from deep to shallow, O. franksi experienced fast photoacclimation and low mortality, and maintained a consistent bacterial community. By contrast, O. annularis experienced high mortality and limited photoacclimation when transplanted from shallow to deep. The photophysiological collapse of O. annularis in the deep environment was associated with an increased microbiome variability and reduction of some bacterial taxa. Differences in the symbiotic algal community were more pronounced between coral species than between depths. Our study suggests that these sibling species are adapted to distinctive light environments partially driven by the algae photoacclimation capacity and the microbiome robustness, highlighting the importance of niche specialization in symbiotic corals for the maintenance of species diversity. Our findings have implications for the management of these threatened Caribbean corals and the effectiveness of coral reef restoration efforts.

Oxybenzone contamination from sunscreen pollution and its ecological threat to Hanauma Bay, Oahu, Hawaii, U.S.A.

Hanauma Bay is a 101-acre bay created by the partial collapse of a volcanic cone and once supported a vibrant coral reef system. It is the most popular swimming area in the Hawaiian Islands and has been reported to have averaged between 2.8 and 3.5 million visitors a year between the 1980s and the 2010s, with visitors averaging between 3000-4000 a day and peaking around 10,000-13,000 per day. Concentrations of oxybenzone and other common UV filters were measured in subsurface water samples and in sands from the beach-shower areas in Hanauma Bay. Results demonstrate that beach showers also can be a source of sunscreen environmental contamination. Hydrodynamic modeling indicates that oxybenzone contamination within Hanauma Bay's waters could be retained between 14 and 50 h from a single release event period. Focusing on only oxybenzone, two different Hazard and Risk Assessment analyses were conducted to determine the danger of oxybenzone to Hanauma Bay's coral reef system. Results indicate that oxybenzone contamination poses a significant threat to the wildlife of Hanauma Bay. To recover Hanauma Bay's natural resources to a healthy condition and to satisfactorily conserve its coral reef and sea grass habitats, effective tourism management policies need to be implemented that mitigate the threat of sunscreen pollution.

Trace element proxies and stable isotopes used to identify water quality threats to elkhorn coral (Acropora palmata) at two national parks in St. Croix, USVI.

Biological impairments have been documented on reefs at two national parks in St. Croix, USVI. Although several water quality parameters have been out of compliance with USVI criteria, whether these parameters or other pollutants are responsible for coral health impacts is unknown. Trace elements quantified in sediment showed four sites at SARI, which is closer than BUIS to settlements and land-derived anthropogenic outflows, had Cu mass fractions above sediment quality guidelines for invertebrate toxicity. Trace elements were also analyzed in the skeleton of threatened elkhorn coral, Acropora palmata, to evaluate potential exposure. Heavy metals (Pb, Zn) were significantly greater in coral skeleton at SARI than BUIS. Cu, Pb, and Zn may be impacting coral health in these parks. Potential anthropogenic sources of these metals were revealed by the coral tissue stable isotope levels (δ13C and δ15N). These findings provide a framework for determining heavy metal impacts on these invaluable reefs.

Morphological, elemental, and boron isotopic insights into pathophysiology of diseased coral growth anomalies.

Coral growth anomalies (GAs) are tumor-like lesions that are detrimental to colony fitness and are commonly associated with high human population density, yet little is known about the disease pathology or calcification behavior. SEM imagery, skeletal trace elements and boron isotopes (δ11B) have been combined as a novel approach to study coral disease. Low Mg/Ca, and high U/Ca, Mo/Ca, and V/Ca potentially suggest a decreased abundance of "centers of calcification" and nitrogen-fixation in GAs. Estimates of carbonate system parameters from δ11B and B/Ca measurements indicate reduced pH (-0.05 units) and [CO32-] within GA calcifying fluid. We theorize GAs re-allocate resources away from internal pH upregulation to sustain elevated tissue growth, resulting in a porous and fragile skeleton. Our findings show that dystrophic calcification processes could explain structural differences seen in GA skeletons and highlight the use of skeletal geochemistry to shed light on disease pathophysiology in corals.

Effect of Louisiana sweet crude oil on a Pacific coral, Pocillopora damicornis.

Recent oil spill responses such as the Deepwater Horizon event have underscored the need for crude oil ecotoxicological threshold data for shallow water corals to assist in natural resource damage assessments. We determined the toxicity of a mechanically agitated oil-seawater mixture (high-energy water-accommodated fraction, HEWAF) of a sweet crude oil on a branched stony coral, Pocillopora damicornis. We report the results of two experiments: a 96 h static renewal exposure experiment and a "pulse-chase" experiment of three short-term exposure durations followed by a recovery period in artificial seawater. Five endpoints were used to determine ecotoxicological values: 1) algal symbiont chlorophyll fluorescence, 2) a tissue regeneration assay and a visual health metric with three endpoints: 3) tissue integrity, 4) tissue color, and 5) polyp behavior. The sum of 50 entrained polycyclic aromatic hydrocarbons (tPAH50) was used as a proxy for oil exposure. For the 96 h exposure dose response experiment, dark-adapted maximum quantum yield (Fv/Fm) of the dinoflagellate symbionts was least affected by crude oil (EC50 = 913 µg/L tPAH50); light-adapted effective quantum yield (EQY) was more sensitive (EC50 =  428 µg/L tPAH50). In the health assessment, polyp behavior (EC50 = 27 µg/L tPAH50) was more sensitive than tissue integrity (EC50 = 806 µg/L tPAH50) or tissue color (EC50 = 926 µg/L tPAH50). Tissue regeneration proved to be a particularly sensitive measurement for toxicity effects (EC50 = 10 µg/L tPAH50). Short duration (6-24 h) exposures using 503 µg/L tPAH50 (average concentration) resulted in negative impacts to P. damicornis and its symbionts. Recovery of chlorophyll a fluorescence levels for 6-24 h oil exposures was observed in a few hours (Fv/Fm) to several days (EQY) following recovery in fresh seawater. The coral health assessments for tissue integrity and tissue color were not affected following short-term oil exposure durations, but the 96 h treatment duration resulted in significant decreases for both. A reduction in polyp behavior (extension) was observed for all treatment durations, with recovery observed for the short-term (6-24 h) exposures within 1-2 days following placement in fresh seawater. Wounded and intact fragments exposed to oil treatments were particularly sensitive, with significant delays observed in tissue regeneration. Estimating ecotoxicological values for P. damicornis exposed to crude oil HEWAFs provides a basis for natural resource damage assessments for oil spills in reef ecosystems. These data, when combined with ecotoxicological values for other coral reef species, will contribute to the development of species sensitivity models.

Expression and Characterization of a Bright Far-red Fluorescent Protein from the Pink-Pigmented Tissues of Porites lobata.

Members of the anthozoan green fluorescent protein (GFP) family display a diversity of photo-physical properties that can be associated with normal and damaged coral tissues. Poritid coral species often exhibit localized pink pigmentation in diseased or damaged tissues. Our spectral and histological analyses of pink-pigmented Porites lobata lesions show co-localization of bright red fluorescence with putative amoebocytes concentrating in the epidermis, suggesting an activated innate immune response. Here we report the cloning, expression, and characterization of a novel red fluorescent protein (plobRFP) from the pink-pigmented tissues associated with lesions on Porites lobata. In vitro, the recombinant plobRFP exhibits a distinct red emission signal of 614 nm (excitation maximum: 578 nm), making plobRFP the furthest red-shifted natural fluorescent protein isolated from a scleractinian coral. The recombinant protein has a high molar extinction coefficient (84,000 M-1 cm-1) and quantum yield (0.74), conferring a notable brightness to plobRFP. Sequence analysis suggests the distinct brightness and marked red shift may be inherent features of plobRFP's chromophore conformation. While plobRFP displays a tendency to aggregate, its high pH stability, photostability, and spectral properties make it a candidate for cell imaging applications and a potential template for engineering optimized RFPs. The association of plobRFP with a possible immune response furthers its potential use as a visual diagnostic and molecular biomarker for monitoring coral health.

Evaluation of Sample Preparation Methods for the Analysis of Reef-Building Corals Using ¹H-NMR-Based Metabolomics.

The field of metabolomics generally lacks standardized methods for the preparation of samples prior to analysis. This is especially true for metabolomics of reef-building corals, where the handful of studies that were published employ a range of sample preparation protocols. The utilization of metabolomics may prove essential in understanding coral biology in the face of increasing environmental threats, and an optimized method for preparing coral samples for metabolomics analysis would aid this cause. The current study evaluates three important steps during sample processing of stony corals: (i) metabolite extraction, (ii) metabolism preservation, and (iii) subsampling. Results indicate that a modified Bligh and Dyer extraction is more reproducible across multiple coral species compared to methyl tert-butyl ether and methanol extractions, while a methanol extraction is superior for feature detection. Additionally, few differences were detected between spectra from frozen or lyophilized coral samples. Finally, extraction of entire coral nubbins increased feature detection, but decreased throughput and was more susceptible to subsampling error compared to a novel tissue powder subsampling method. Overall, we recommend the use of a modified Bligh and Dyer extraction, lyophilized samples, and the analysis of brushed tissue powder for the preparation of reef-building coral samples for ¹H NMR metabolomics.

Empty Niches after Extinctions Increase Population Sizes of Modern Corals.

Large environmental fluctuations often cause mass extinctions, extirpating species and transforming communities [1, 2]. While the effects on community structure are evident in the fossil record, demographic consequences for populations of individual species are harder to evaluate because fossils reveal relative, but not absolute, abundances. However, genomic analyses of living species that have survived a mass extinction event offer the potential for understanding the demographic effects of such environmental fluctuations on extant species. Here, we show how environmental variation since the Pliocene has shaped demographic changes in extant corals of the genus Orbicella, major extant reef builders in the Caribbean that today are endangered. We use genomic approaches to estimate previously unknown current and past population sizes over the last 3 million years. Populations of all three Orbicella declined around 2-1 million years ago, coincident with the extinction of at least 50% of Caribbean coral species. The estimated changes in population size are consistent across the three species despite their ecological differences. Subsequently, two shallow-water specialists expanded their population sizes at least 2-fold, over a time that overlaps with the disappearance of their sister competitor species O. nancyi (the organ-pipe Orbicella). Our study suggests that populations of Orbicella species are capable of rebounding from reductions in population size under suitable conditions and that the effective population size of modern corals provides rich standing genetic variation for corals to adapt to climate change. For conservation genetics, our study suggests the need to evaluate genetic variation under appropriate demographic models.

Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands.

Benzophenone-3 (BP-3; oxybenzone) is an ingredient in sunscreen lotions and personal-care products that protects against the damaging effects of ultraviolet light. Oxybenzone is an emerging contaminant of concern in marine environments­produced by swimmers and municipal, residential, and boat/ship wastewater discharges. We examined the effects of oxybenzone on the larval form (planula) of the coral Stylophora pistillata, as well as its toxicity in vitro to coral cells from this and six other coral species. Oxybenzone is a photo-toxicant; adverse effects are exacerbated in the light. Whether in darkness or light, oxybenzone transformed planulae from a motile state to a deformed, sessile condition. Planulae exhibited an increasing rate of coral bleaching in response to increasing concentrations of oxybenzone. Oxybenzone is a genotoxicant to corals, exhibiting a positive relationship between DNA-AP lesions and increasing oxybenzone concentrations. Oxybenzone is a skeletal endocrine disruptor; it induced ossification of the planula, encasing the entire planula in its own skeleton. The LC50 of planulae exposed to oxybenzone in the light for an 8- and 24-h exposure was 3.1 mg/L and 139 µg/L, respectively. The LC50s for oxybenzone in darkness for the same time points were 16.8 mg/L and 779 µg/L. Deformity EC20 levels (24 h) of planulae exposed to oxybenzone were 6.5 µg/L in the light and 10 µg/L in darkness. Coral cell LC50s (4 h, in the light) for 7 different coral species ranges from 8 to 340 µg/L, whereas LC20s (4 h, in the light) for the same species ranges from 0.062 to 8 µg/L. Coral reef contamination of oxybenzone in the U.S. Virgin Islands ranged from 75 µg/L to 1.4 mg/L, whereas Hawaiian sites were contaminated between 0.8 and 19.2 µg/L. Oxybenzone poses a hazard to coral reef conservation and threatens the resiliency of coral reefs to climate change.

Toxicological effects of the sunscreen UV filter, benzophenone-2, on planulae and in vitro cells of the coral, Stylophora pistillata.

Benzophenone-2 (BP-2) is an additive to personal-care products and commercial solutions that protects against the damaging effects of ultraviolet light. BP-2 is an "emerging contaminant of concern" that is often released as a pollutant through municipal and boat/ship wastewater discharges and landfill leachates, as well as through residential septic fields and unmanaged cesspits. Although BP-2 may be a contaminant on coral reefs, its environmental toxicity to reefs is unknown. This poses a potential management issue, since BP-2 is a known endocrine disruptor as well as a weak genotoxicant. We examined the effects of BP-2 on the larval form (planula) of the coral, Stylophora pistillata, as well as its toxicity to in vitro coral cells. BP-2 is a photo-toxicant; adverse effects are exacerbated in the light versus in darkness. Whether in darkness or light, BP-2 induced coral planulae to transform from a motile planktonic state to a deformed, sessile condition. Planulae exhibited an increasing rate of coral bleaching in response to increasing concentrations of BP-2. BP-2 is a genotoxicant to corals, exhibiting a strong positive relationship between DNA-AP lesions and increasing BP-2 concentrations. BP-2 exposure in the light induced extensive necrosis in both the epidermis and gastro dermis. In contrast, BP-2 exposure in darkness induced autophagy and autophagic cell death.The LC50 of BP-2 in the light for an 8 and 24 hour exposure was 120 parts per million (ppm) and 165 parts per billion (ppb), respectively. The LC50s for BP-2 in darkness for the same time points were 144 parts per million and 548 parts per billion [corrected].

Heat-stress and light-stress induce different cellular pathologies in the symbiotic dinoflagellate during coral bleaching.

Coral bleaching is a significant contributor to the worldwide degradation of coral reefs and is indicative of the termination of symbiosis between the coral host and its symbiotic algae (dinoflagellate; Symbiodinium sp. complex), usually by expulsion or xenophagy (symbiophagy) of its dinoflagellates. Herein, we provide evidence that during the earliest stages of environmentally induced bleaching, heat stress and light stress generate distinctly different pathomorphological changes in the chloroplasts, while a combined heat- and light-stress exposure induces both pathomorphologies; suggesting that these stressors act on the dinoflagellate by different mechanisms. Within the first 48 hours of a heat stress (32°C) under low-light conditions, heat stress induced decomposition of thylakoid structures before observation of extensive oxidative damage; thus it is the disorganization of the thylakoids that creates the conditions allowing photo-oxidative-stress. Conversely, during the first 48 hours of a light stress (2007 µmoles m(-2) s(-1) PAR) at 25°C, condensation or fusion of multiple thylakoid lamellae occurred coincidently with levels of oxidative damage products, implying that photo-oxidative stress causes the structural membrane damage within the chloroplasts. Exposure to combined heat- and light-stresses induced both pathomorphologies, confirming that these stressors acted on the dinoflagellate via different mechanisms. Within 72 hours of exposure to heat and/or light stresses, homeostatic processes (e.g., heat-shock protein and anti-oxidant enzyme response) were evident in the remaining intact dinoflagellates, regardless of the initiating stressor. Understanding the sequence of events during bleaching when triggered by different environmental stressors is important for predicting both severity and consequences of coral bleaching.

A survey of environmental pollutants and cellular-stress markers of Porites astreoides at six sites in St. John, U.S. Virgin Islands.

Coral communities along the coast of St. John, U.S. Virgin Islands have exhibited site-specific behavior in declines. In order to determine if these specific coral communities are stressed and whether a pollutant or environmental factor present at this site is a probable stressor, we surveyed six near-shore coral communities in St. John, USVI for environmental pollutants and to determine the cellular physiological condition of the coral, Porites astreoides. The six sites within St. John are Cruz Bay, Caneel Bay, Hawksnest Bay, Trunk Bay, Tektite Reef in Beehive Bay, and Red Point. Red Point was considered the reference site because of its abundance and diversity of species, and it was the furthest removed from down-stream and down-current anthropogenic activities. All sites showed distinct cellular-stress marker patterns, indicating that the physiological condition of each population was different. Populations at Cruz, Hawksnest, Trunk, and Tektite were stressed, as indicated by high levels of DNA lesions and expression of stress proteins. Hawksnest and Tektite were contaminated with polyaromatic hydrocarbons (PAHs), while Cruz was contaminated with semi-volatile organochlorines and nitrogen-based biocides. At least for Hawksnest and Tektite, stress-marker patterns were consistent with an exposure to PAHs. Fecal coliform levels were high in Cruz and Trunk, indicating fecal contamination, as well as consideration for management action. Results from this study serve as a justification for a more thorough and methodical investigation into the stressors responsible for declines of coral populations within St. John. Furthermore, this study supports the argument for the importance of local factors contributing to regional coral reef declines; that not all forces impacting coral are global.

Threatened corals provide underexplored microbial habitats.

Contemporary in-depth sequencing of environmental samples has provided novel insights into microbial community structures, revealing that their diversity had been previously underestimated. Communities in marine environments are commonly composed of a few dominant taxa and a high number of taxonomically diverse, low-abundance organisms. However, studying the roles and genomic information of these "rare" organisms remains challenging, because little is known about their ecological niches and the environmental conditions to which they respond. Given the current threat to coral reef ecosystems, we investigated the potential of corals to provide highly specialized habitats for bacterial taxa including those that are rarely detected or absent in surrounding reef waters. The analysis of more than 350,000 small subunit ribosomal RNA (16S rRNA) sequence tags and almost 2,000 nearly full-length 16S rRNA gene sequences revealed that rare seawater biosphere members are highly abundant or even dominant in diverse Caribbean corals. Closely related corals (in the same genus/family) harbored similar bacterial communities. At higher taxonomic levels, however, the similarities of these communities did not correlate with the phylogenetic relationships among corals, opening novel questions about the evolutionary stability of coral-microbial associations. Large proportions of OTUs (28.7-49.1%) were unique to the coral species of origin. Analysis of the most dominant ribotypes suggests that many uncovered bacterial taxa exist in coral habitats and await future exploration. Our results indicate that coral species, and by extension other animal hosts, act as specialized habitats of otherwise rare microbes in marine ecosystems. Here, deep sequencing provided insights into coral microbiota at an unparalleled resolution and revealed that corals harbor many bacterial taxa previously not known. Given that two of the coral species investigated are listed as threatened under the U.S. Endangered Species Act, our results add an important microbial diversity-based perspective to the significance of conserving coral reefs.

Cellular pathology and histopathology of hypo-salinity exposure on the coral Stylophora pistillata.

Coral reefs can experience extreme salinity changes, particularly hypo-salinity, as a result of storms, heavy rainy seasons (e.g., monsoons), and coastal runoff. Field and laboratory observations have documented that corals exposed to hypo-saline conditions can undergo extensive bleaching and mortality. There is controversy in the literature as to whether hypo-saline conditions induce a pathological response in corals, and if there is a relationship between decreasing salinity treatment and pathological responses. To test the hypothesis that hypo-salinity exposure does not have a pathological effect on coral, we used histological and cellular diagnostic methods to characterize the pathology in hypo-salinity-exposed corals. Colonies of Stylophora pistillata were exposed to five salinity concentrations [39 parts per thousand (ppt), 32 ppt, 28 ppt, 24 ppt, and 20 ppt] that may realistically occur on a reef. Histological examination indicated an increasing severity of pathomorphologies associated with decreasing salinity, including increased tissue swelling, degradation and loss of zooxanthellae, and tissue necrosis. Pulse-amplitude modulated chlorophyll fluorimetry kinetics demonstrated a decreasing photosynthetic efficiency with decreasing salinity conditions. Cytochrome P450 levels were affected by even slight changes in salinity concentration suggesting that detoxification pathways, as well as several endocrine pathways, may be adversely affected. Finally, these studies demonstrated that hypo-saline conditions can induce an oxidative-stress response in both the host and in its algal symbiont, and in so doing, may synergistically increase oxidative-stress burdens. As with other types of environmental stresses, exposure to hypo-saline conditions may have long-term consequences on coral physiology.

Symbiophagy as a cellular mechanism for coral bleaching.

Coral bleaching is a major contributor to the global declines of coral reefs. This phenomenon is characterized by the loss of symbiotic algae, their pigments or both. Despite wide scientific interest, the mechanisms by which bleaching occurs are still poorly understood. Here we report that the removal of the symbiont during light and temperature stress is achieved using the host's cellular autophagic-associated machinery. Host cellular and subcellular morphologies showed increased vacuolization and appearance of autophagic membranes surrounding a variety of organelles and surrounding the symbiotic algae. Markers of autophagy (Rab 7 and LAS) corroborate these observations. Results showed that during stress the symbiont vacuolar membrane is transformed from a conduit of nutrient exchange to a digestive organelle resulting in the consumption of the symbiont, a process we term symbiophagy. We posit that during a stress event, the mechanism maintaining symbiosis is destabilized and symbiophagy is activated, ultimately resulting in the phenomenon of bleaching. Symbiophagy may have evolved from a more general primordial innate intracellular protective pathway termed xenophagy.