Differential molecular biomarker expression in corals over a gradient of water quality stressors in Maunalua Bay, Hawaii.

Coral reefs globally face unprecedented challenges from anthropogenic stressors, necessitating innovative approaches for effective assessment and management. Molecular biomarkers, particularly those related to protein expressions, provide a promising avenue for diagnosing coral health at the cellular level. This study employed enzyme-linked immunosorbent assays to evaluate stress responses in the coral Porites lobata along an environmental gradient in Maunalua Bay, Hawaii. The results revealed distinct protein expression patterns correlating with anthropogenic stressor levels across the bay. Some proteins, such as ubiquitin and Hsp70, emerged as sensitive biomarkers, displaying a linear decrease in response along the environmental gradient, emphasizing their potential as indicators of stress. Our findings highlighted the feasibility of using protein biomarkers for real-time assessment of coral health and the identification of stressors. The identified biomarkers can aid in establishing stress thresholds and evaluating the efficacy of management interventions. Additionally, we assessed sediment and water quality from the inshore areas in the bay and identified organic contaminants, including polycyclic aromatic hydrocarbons and pesticides, in bay sediments and waters.

Environmental Concentrations of Herbicide Prometryn Render Stress-Tolerant Corals Susceptible to Ocean Warming.

Global warming has caused the degradation of coral reefs around the world. While stress-tolerant corals have demonstrated the ability to acclimatize to ocean warming, it remains unclear whether they can sustain their thermal resilience when superimposed with other coastal environmental stressors. We report the combined impacts of a photosystem II (PSII) herbicide, prometryn, and ocean warming on the stress-tolerant coral Galaxea fascicularis through physiological and omics analyses. The results demonstrate that the heat-stress-induced inhibition of photosynthetic efficiency in G. fascicularis is exacerbated in the presence of prometryn. Transcriptomics and metabolomics analyses indicate that the prometryn exposure may overwhelm the photosystem repair mechanism in stress-tolerant corals, thereby compromising their capacity for thermal acclimation. Moreover, prometryn might amplify the adverse effects of heat stress on key energy and nutrient metabolism pathways and induce a stronger response to oxidative stress in stress-tolerant corals. The findings indicate that the presence of prometryn at environmentally relevant concentrations would render corals more susceptible to heat stress and exacerbate the breakdown of coral Symbiodiniaceae symbiosis. The present study provides valuable insights into the necessity of prioritizing PSII herbicide pollution reduction in coral reef protection efforts while mitigating the effects of climate change.

A combined proteomics and metabolomics analysis reveals the invisible regulation of plant root responses to oxybenzone (benzophenone-3) stress.

Oxybenzone, an environmental pollutant affecting both agriculture and aquatic ecological integrity, has been demonstrated to act as a physiological and metabolic inhibitor on plants, animals, and microorganisms. Research on oxybenzone in higher plants has focused on the above-ground anatomy (leaves), while research on the under-ground parts (roots) has been neglected. In this study, the changes in plant root protein expression and metabolic pathways under oxybenzone treatment were explored through a combined proteomics and metabolomics analysis. A total of 506 differential proteins and 96 differential metabolites were identified, which were mainly distributed in critical pathways such as those for carbon (C) and nitrogen (N) metabolism, lipid metabolism, and antioxidation. Bioinformatics analysis shows that oxybenzone toxicity is predominantly reflected in alterations to root respiratory homeostasis and the manifestation of damaging reactive oxygen species (ROS) and membrane lipid peroxidation, changes to disease resistance-associated proteins, changes to normal C-flow distribution, and the inhibition of cell absorption and utilization of N sources. Plants respond to oxybenzone stress mainly by reconfiguring the mitochondrial electron-transport-chain to bypass oxidative-damage components; improving the efficiency of the antioxidant system to remove excessively accumulated ROS; promoting the detoxification of harmful membrane lipid peroxides; increasing osmotic adjustment substance (such as proline and raffinose) accumulation; adjusting C flow distribution to produce more nicotinamide adenine dinucleotide phosphate (NADPH) for the glutathione cycle; and accumulating free amino acids to increase plant stress tolerant. Our results are the first to map the changes in the physiological and metabolic regulatory network of higher plant roots under oxybenzone stress.

Proteomics and Lysine Acetylation Modification Reveal the Responses of Pakchoi (Brassica rapa L. ssp. chinensis) to Oxybenzone Stress.

The broad-spectrum UV filter oxybenzone is toxic to plants at environmentally relevant concentrations. Lysine acetylation (LysAc) is one of the essential post-translational modifications (PTMs) in plant signaling responses. The goal of this study was to uncover the LysAc regulatory mechanism in response to toxic exposures to oxybenzone as a first step in elucidating xenobiotic acclimatory reactions by using the model Brassica rapa L. ssp. chinensis. A total of 6124 sites on 2497 proteins were acetylated, 63 proteins were differentially abundant, and 162 proteins were differentially acetylated under oxybenzone treatment. Bioinformatics analysis showed that a large number of antioxidant proteins were significantly acetylated under oxybenzone treatment, implying that LysAc alleviated the adverse effects of reactive oxygen species (ROS) by inducing antioxidant systems and stress-related proteins; the significant changes in acetylation modification of enzymes involved in different branches of carbon metabolism in plants under oxybenzone treatment mean that plants can change the direction of carbon flow allocation by regulating the activities of carbon metabolism-related enzymes. Our results profile the protein LysAc under oxybenzone treatment and propose an adaptive mechanism at the post-translational level of vascular plants in response to pollutants, providing a dataset reference for future related research.

Comparison of toxicological effects of oxybenzone, avobenzone, octocrylene, and octinoxate sunscreen ingredients on cucumber plants (Cucumis sativus L.).

Oxybenzone (OBZ), avobenzone (AVB), octocrylene (OCR) and octinoxate (OMC) are ultraviolet (UV) filters commonly added to chemical sunscreens. These UV filters are known to widely contaminate the environment through a variety of anthropogenic sources, including sewage discharge. However, systematic studies of the damage caused by these four UV filters and their toxicopathological differences in a variety of plant species are lacking. In this study, we demonstrated that irrigation with water containing these four UV filters could significantly inhibit the aboveground growth of cucumber plant. All of the UV filters decreased photosynthesis through nonstomatal factors but via different inhibitory mechanisms. Only OBZ inhibited photosynthesis by directly inhibiting photosynthetic electron transport, while the other three (AVB, OCR, and OMC) inhibited photosynthesis by inhibiting the Calvin-Benson cycle. Additionally, these four UV filters also decreased plant respiration under long-term treatment. Photosynthesis and respiration inhibition led to the over production of reactive oxygen species (ROS) and the formation of lipid peroxidation damage products, which further damaged the structure and function of plant cells, causing secondary pathologies and potentially leading to reduced crop yields. The study also demonstrated that these four UV filters caused different degrees of phototoxic damage to cucumber plants. On the basis of comprehensive evaluation, we speculated that the order of the four UV filters in terms of plant damage was OBZ > AVB > OMC > OCR. Because of the severe damaging effects of these UV filters on plant growth, the application of contaminated biosolids/reclaimed water in agriculture reduces agricultural production and may damage ecosystems. The results of this study can advance recognition of the hazards associated with environmental and agricultural pollution via UV filters and encourage consumers and the industry to limit or reduce the application of cosmetics and over-the-counter drugs containing these substances.

The toxicological effects of oxybenzone, an active ingredient in suncream personal care products, on prokaryotic alga Arthrospira sp. and eukaryotic alga Chlorella sp.

Oxybenzone (OBZ; benzophenone-3, CAS# 131-57-7) is a known pollutant of aquatic and marine ecosystems, and is an ingredient in over 3000 personal care products, as well as many types of plastics. The aim of this study is to explore the different toxicities of OBZ on an eukaryotic (Chlorella sp.) and a prokaryotic algae (Arthrospira sp.). OBZ is a photo-toxicant, with all observed toxicities more sever in the light than in the dark. Cell growth and chlorophyll inhibition were positively correlated with increasing OBZ concentrations over time. Twenty days treatment with OBZ, as low as 22.8 ng L-1, significantly inhibited the growth and chlorophyll synthesis of both algae. Both algae were noticeably photo-bleached after 7 days of exposure to OBZ concentrations higher than 2.28 mg L-1. Relatively low OBZ concentrations (0.228 mg L-1) statistically constrained photosynthetic and respiratory rates via directly inhibiting photosynthetic electron transport (PET) and respiration electron transport (RET) mechanisms, resulting in over production of reactive oxygen species (ROS). Transmission and scanning electron microscopy showed that the photosynthetic and respiratory membrane structures were damaged by OBZ exposure in both algae. Additionally, PET inhibition suppressed ATP production for CO2 assimilation via the Calvin-Benson cycle, further limiting synthesis of other biomacromolecules. RET restriction limited ATP generation, restricting the energy supply used for various life activities in the cell. These processes further impacted on photosynthesis, respiration and algal growth, representing secondary OBZ-induced algal damages. The data contained herein, as well as other studies, supports the argument that global pelagic and aquatic phytoplankton could be negatively influenced by OBZ pollution.

Can oxybenzone cause Hirschsprung's disease?

Oxybenzone is a ultraviolet (UV) absorber used in 70% of sunscreen products, is a recognized endocrine disrupting chemical (EDC) and is small enough to pass through skin and placenta barriers. Numerous studies have identified this chemical in the urine/blood of pregnant women as well as in fetal and umbilical cord blood. A recent study demonstrated that women with medium to high levels of oxybenzone in their urine was associated with giving birth to neonates with Hirschsprung's Disease (HSCR). Testing in human cell lines confirmed that low levels of oxybenzone has the potential to disrupt cell migration and function in a manner similar to what is associated with HSCR. Analysis of human exposure levels to oxybenzone from sunscreen use, under normal conditions, demonstrates that enough chemical can cross into the mother's blood making it available to the fetus at high enough levels that can indeed inhibit migration of neural crest cells during critical embryonic development.

Dermatological and environmental toxicological impact of the sunscreen ingredient oxybenzone/benzophenone-3.

Oxybenzone (Benzophenone-3) is an emerging human and environmental contaminant used in sunscreens and personal care products to help minimize the damaging effects of ultraviolet radiation. The Center for Disease Control fourth national report on human exposure to environmental chemicals demonstrated that approximately 97% of the people tested have oxybenzone present in their urine, and independent scientists have reported various concentrations in waterways and fish worldwide. Oxybenzone can also react with chlorine, producing hazardous by-products that can concentrate in swimming pools and wastewater treatment plants. Moreover, adverse reactions could very well be increased by the closed loop of ingesting fish contaminated with oxybenzone and/or washing the ingredient off our bodies and having it return in drinking water as treatment plants do not effectively remove the chemical as part of their processing protocols. In humans, oxybenzone has been reported to produce contact and photocontact allergy reactions, implemented as a possible endocrine disruptor and has been linked to Hirschsprung's disease. Environmentally, oxybenzone has been shown to produce a variety of toxic reactions in coral and fish ranging from reef bleaching to mortality. Lastly, with the rise in skin cancer rates and the availability of more effective sunscreen actives such as micronized zinc oxide and titanium dioxide, serious doubts about the relative prevention benefit of personal care products containing oxybenzone must be raised and compared with the potential negative health and environmental effects caused by the accumulation of this and other chemicals in the ecosystem.

Migration of nonylphenol from food-grade plastic is toxic to the coral reef fish species Pseudochromis fridmani.

Nonylphenol (NP) is a non-ionic surfactant used extensively in industrial applications, personal care products, and many plastics. We exposed marine orchid dottybacks (Pseudochromis fridmani) for 48h to either glass, Teflon, or two bags labeled as FDA food-grade polyethylene (PE1 and PE2) from different manufacturers. The PE2 bags leached high levels of NP into the contact water, which were taken up by the fish, and decreased short and long-term survival. Concentrations of NP that leached from the bags were consistent with 96h LC50 values determined in this study, indicating NP is the likely toxic agent. Despite being similarly labeled, the NP concentrations that leached from the bags and the resultant toxicity to the fish varied dramatically between manufacturers. This study highlights that some plastics, labeled as food-safe, can be highly toxic to aquatic animals, and could pose a greater threat to humans than previously realized. This study also highlights risks for aquatic animals exposed to increasing quantities of plastic waste.

The use of cellular diagnostics for identifying sub-lethal stress in reef corals.

Coral reefs throughout the world are exhibiting documented declines in coral cover and species diversity, which have been linked to anthropogenic stressors including land-based sources of pollution. Reductions in coastal water and substratum quality are affecting coral survivorship, reproduction and recruitment, and hence, the persistence of coral reefs. One major obstacle in effectively addressing these declines is the lack of tools that can identify cause-and-effect relationships between stressors and specific coral reef losses, while a second problem is the inability to measure the efficacy of mitigation efforts in a timely fashion. We examined corals from six coral reefs on Guam, Mariana Islands, which were being affected by different environmental stressors (e.g. PAH's, pesticides, PCB's and sedimentation). Cellular diagnostic analysis differentiated the cellular-physiological condition of these corals. Examination of protein expression provided insight into their homeostatic responses to chemical and physical stressors in exposed corals prior to outright mortality, providing improved opportunities for developing locally-based management responses. This approach adds critically needed tools for addressing the effects of multiple stressors on corals and will allow researchers to move beyond present assessment and monitoring techniques that simply document the loss of coral abundance and diversity.

Concentrations of Irgarol in selected marinas of Oahu, Hawaii and effects on settlement of coral larval.

This study examined concentrations of Irgarol 1051(®) in selected marinas on the island of Oahu, Hawaii and used laboratory bioassays to assess effects of Irgarol on coral larval settlement. Field surveys of small boat marinas performed in 2006-2007 revealed low concentrations of Irgarol 1051(®), an antifouling paint additive, ranging from non-detected (<17 ng/l) to 283 ng/l. The highest concentrations of Irgarol 1051(®) were found in marinas with low flushing rates and a high density of moored boats and boat traffic. The potential effect of Irgarol 1051(®) on coral larval settlement was evaluated in the laboratory using planulae from Porites hawaiiensis, a zooxanthellate shade-dwelling coral found in Hawaiian waters. Exposure to Irgarol 1051(®) at 100 ng/l resulted in a statistically significant reduction in settlement of coral larvae. This was within the range of Irgarol 1051(®) concentrations found in some of the marinas surveyed on the island of Oahu but Irgarol was not detected in seawater samples at offshore reefs.

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.

A novel method for coral explant culture and micropropagation.

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

In vitro cell-toxicity screening as an alternative animal model for coral toxicology: effects of heat stress, sulfide, rotenone, cyanide, and cuprous oxide on cell viability and mitochondrial function.

The logistics involved in obtaining and maintaining large numbers of corals hampers research on the toxicological effects of environmental contaminants for this ecologically and economically important taxon. A method for creating and culturing single-cell suspensions of viable coral cells was developed. Cell segregation/separation was based on specific cell densities and resulting cell cultures were viable for at least 2 mos. Low-density cells lacking symbiotic zooxanthallae and rich in mitochondria were isolated and cultured for toxicity studies. Cells were exposed to differing degrees or concentrations of heat stress, rotenone, cyanide, sulfide, and cuprous oxide. Cells were assayed for mitochondrial membrane potential using the fluorescent probe, JC-9, and for overall viability using the MTT/formazan spectrophotometric viability assay. Significant differences were observed between controls and treatments and the efficacy of this method was validated; only 2 cm(2) of tissue was required for a seven-point concentration-exposure series.

Assessment of cellular and functional biomarkers in bivalves exposed to ecologically relevant abiotic stressors.

An understanding of the complex effects of the environment on biomarkers of bivalve health is essential for aquaculturists to successfully select field culture sites and monitor bivalve health in these sites and in hatcheries. We tested several whole-organism (functional) and cellular-level biomarkers as indicators of health of the cultured, stress-tolerant northern quahog (hard clam) Mercenaria mercenaria. We performed single- and dual-stressor experiments that were consistent with available water quality data from a clam culture area on the Gulf coast of Florida. Clams from the culture area were exposed over a 14-d period to low O2 (hypoxia), elevated temperature, hyposalinity, and a combination of elevated temperature and hyposalinity. There was no clear relationship between the functional and cellular-level biomarkers, with most of the treatment effects being detected at the whole-organism level but not the cellular level. Survival and burial ability were significantly affected by elevated temperature and by the combination of elevated temperature and hyposalinity. Glycogen content decreased over the experiment duration and did not differ significantly among treatments. There were no significant changes in expression patterns of eight stress proteins or in the levels of oxidatively damaged RNA. The results highlight the importance of investigating the effects of multiple stressors in short-term, controlled laboratory conditions and suggest that such cellular-level biomarker assays should be paired with functional biomarkers to better understand the responses of highly stress-tolerant species.

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.

Increased expression of stress proteins in the surf clam Donax variabilis following hydrogen sulfide exposure.

Endogenous free radical production and resulting oxidative damage may result from exposure to hypoxia, hyperoxia, or hydrogen sulfide. Previous investigations of sulfide-induced oxidative damage have produced conflicting results, perhaps because these studies utilized species presumably adapted to sulfide. We examined the effects of sulfide, hypoxia and hyperoxia on the surf clam Donax variabilis to test whether these stressors induce a cellular response to oxidative stress. These clams inhabit high-energy sandy beaches and are unlikely to have specific adaptations to these stressors. In duplicate flow-through experiments performed in fall and spring, clams were exposed to normoxia (22 kPa P(O(2))), hypoxia (10 kPa), hyperoxia (37 kPa), or sulfide with normoxia ( approximately 100 mumol L(-1), 22 kPa respectively) for 24 h. We quantified whole-animal expression of three antioxidants (Cu/Zn and Mn superoxide dismutases, glutathione peroxidase), a lipid peroxidation marker (4-hydroxy-2E-nonenol-adducted protein), a DNA repair enzyme (OGG1-m), four heat shock proteins (small Hsp, Hsp60, Hsp70, and mitochondrial Hsp70), ubiquitin, and actin. Clams exposed to sulfide showed upregulation of the greatest number of stress proteins and the pattern was consistent with a cellular response to oxidative stress. Furthermore, there was a marked seasonality, with greater stress protein expression in clams from the spring.

Cellular physiological effects of the MV Kyowa violet fuel-oil spill on the hard coral, Porites lobata.

The grounding of the Merchant Vessel (MV) Kyowa Violet on a coral reef near Yap, Federated States of Micronesia, in December 2002 resulted in the release of an estimated 55,000 to 80,000 gallons of intermediate fuel oil grade 180. The immediate impact was the widespread coating of mangroves and the intertidal zone along more than 8 km of coastline. Of greater concern, however, was the partitioning of the fuel oil in the water column, leading to chronic exposure of organisms in the ecosystem for a considerable period after the initial event. Herein, we report on our examination of one coral species, Porites lobata, nearly three months after the initial exposure. We investigated whether changes in cellular physiology were consistent with the pathological profile that results from the interaction of corals with polycyclic aromatic hydrocarbons, the principal constituent of fuel oil. Specifically, we document, to our knowledge for the first time, changes in the cellular physiological condition of an exposed coral population affected by a fuel-oil spill. We also provide evidence that the observed changes are consistent with a recent exposure to fuel oil, as evidenced by the presence of characteristic cellular lesions attributed to polycyclic aromatic hydrocarbons. Finally, our data support a model for a mechanistic relationship between the cellular pathological profile of the coral and a recent petroleum exposure, such as the MV Kyowa Violet fuel oil spill.