Microbiome depletion and recovery in the sea anemone, Exaiptasia diaphana, following antibiotic exposure.

Microbial species that comprise host-associated microbiomes play an essential role in maintaining and mediating the health of plants and animals. While defining the role of individual or even complex communities is important toward quantifying the effect of the microbiome on host health, it is often challenging to develop causal studies that link microbial populations to changes in host fitness. Here, we investigated the impacts of reduced microbial load following antibiotic exposure on the fitness of the anemone, Exaiptasia diaphana and subsequent recovery of the host's microbiome. Anemones were exposed to two different types of antibiotic solutions for 3 weeks and subsequently held in sterilized seawater for a 3-week recovery period. Our results revealed that both antibiotic treatments reduced the overall microbial load during and up to 1 week post-treatment. The observed reduction in microbial load was coupled with reduced anemone biomass, halted asexual reproduction rates, and for one of the antibiotic treatments, the partial removal of the anemone's algal symbiont. Finally, our amplicon sequencing results of the 16S rRNA gene revealed that anemone bacterial composition only shifted in treated individuals during the recovery phase of the experiment, where we also observed a significant reduction in the overall diversity of the microbial community. Our work implies that the E. diaphana's microbiome contributes to host fitness and that the recovery of the host's microbiome following disturbance with antibiotics leads to a reduced, but stable microbial state.IMPORTANCEExaiptasia diaphana is an emerging model used to define the cellular and molecular mechanisms of coral-algal symbioses. E. diaphana also houses a diverse microbiome, consisting of hundreds of microbial partners with undefined function. Here, we applied antibiotics to quantify the impact of microbiome removal on host fitness as well as define trajectories in microbiome recovery following disturbance. We showed that reduction of the microbiome leads to negative impacts on host fitness, and that the microbiome does not recover to its original composition while held under aseptic conditions. Rather the microbiome becomes less diverse, but more consistent across individuals. Our work is important because it suggests that anemone microbiomes play a role in maintaining host fitness, that they are susceptible to disturbance events, and that it is possible to generate gnotobiotic individuals that can be leveraged in microbiome manipulation studies to investigate the role of individual species on host health.

Antibiotics alter development and gene expression in the model cnidarian Nematostella vectensis.

Background: Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis. Methods: Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones. Results: Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments. Discussion: Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.

The regulatory role of GABAA receptor in Actinia equina nervous system and the possible effect of global ocean acidification.

Global warming and connected acidification of the world ocean attract a substantial amount of research efforts, in particular in a context of their impact on behaviour and metabolism of marine organisms, such as Cnidaria. Nevertheless, mechanisms underlying Cnidarians' neural signalling and behaviour and their (possible) alterations due to the world ocean acidification remain poorly understood. Here we researched for the first time modulation of GABAA receptors (GABAARs) in Actinia equina (Cnidaria: Anthozoa) by pH fluctuations within a range predicted by the world ocean acidification scenarios for the next 80-100 years and by selective pharmacological activation. We found that in line with earlier studies on vertebrates, both changes of pH and activation of GABAARs with a selective allosteric agonist (diazepam) modulate electrical charge transfer through GABAAR and the whole-cell excitability. On top of that, diazepam modifies the animal behavioural reaction on startle response. However, despite behavioural reactions displayed by living animals are controlled by GABAARs, changes of pH do not alter them significantly. Possible mechanisms underlying the species resistance to acidification impact are discussed.

Cnidarian Cell Cryopreservation: A Powerful Tool for Cultivation and Functional Assays.

Cnidarian primary cell cultures have a strong potential to become a universal tool to assess stress-response mechanisms at the cellular level. However, primary cell cultures are time-consuming regarding their establishment and maintenance. Cryopreservation is a commonly used approach to provide stable cell stocks for experiments, but it is yet to be established for Cnidarian cell cultures. The aim of this study was therefore to design a cryopreservation protocol for primary cell cultures of the Cnidarian Anemonia viridis, using dimethyl sulfoxide (DMSO) as a cryoprotectant, enriched or not with fetal bovine serum (FBS). We determined that DMSO 5% with 25% FBS was an efficient cryosolution, resulting in 70% of post-thaw cell survival. The success of this protocol was first confirmed by a constant post-thaw survival independently of the cell culture age (up to 45 days old) and the storage period (up to 87 days). Finally, cryopreserved cells displayed a long-term recovery with a maintenance of the primary cell culture parameters and cellular functions: formation of cell aggregates, high viability and constant cell growth, and unchanged intrinsic resistance to hyperthermal stress. These results will further bring new opportunities for the scientific community interested in molecular, cellular, and biochemical aspects of cnidarian biology.

Plastic pellets trigger feeding responses in sea anemones.

Multiple mechanisms for plastic consumption by marine animals have been proposed based on the feeding cues and behavior of the animal studied. We investigated plastic consumption in sea anemones. We found that anemones readily consumed pristine National Institute of Standards and Technology low-density polyethylene and high-density polyethylene II and III pre-production pellets. Anemone weight, crown area, and number of tentacles were measured before and after 12 days of daily pellet consumption. Crown area significantly increased for control anemones only. Fresh anemones were then sequentially fed consumed and egested pellets from two of the earlier daily trials to measure feeding retention time, which decreased over three to four feedings. The concentrations of elements in anemones (zinc, iron, arsenic, manganese, chromium, copper, vanadium, selenium, nickel, cadmium, and cobalt) were similar to control anemones that were not exposed to pellets. Lead concentrations were significantly higher in anemones fed HDPE III pellets as compared to control. Plastic consumption by marine animals might be reduced by reducing the amount of plastic that enters the ocean and understanding the chemical triggers underlying plastic consumption.

Heavy Metal Bioaccumulation in the Anemone Paraphelliactis pabista Dunn, 1982 (Actiniaria: Hormathiidae) from the Hydrothermal System of Guaymas Basin, Gulf of California.

A single specimen of the anemone Paraphelliactis pabista was recovered from the Southern Trough of Guaymas Basin during the deep-sea expedition Extreme 2008 conducted onboard the R/V Atlantis/DSRV-2 ALVIN. We studied the bioaccumulation capacity of heavy metals in various tissues of the anemone (oral disk-columella-pedal disk), and retention or adhesion of mineral particles in the epidermis, mesoglea, and gastrodermis. The digested tissues were analyzed for As, Ba, Co, Cu, Cr, Fe, Mn, Ni, Pb, Se, Sb, Sr, Ti, V, and Zn by inductively coupled plasma mass spectrometry. This analysis revealed the capacity of P. pabista for accumulating heavy metals. The predominant mineral particles identified in tissue samples was barite followed by Fe, aluminum-silicates, Sr, and with less presence Cr, Ti, and pyrite. Of the three body compartments analyzed of this anemone, the oral and pedal disks show a greater capacity of bioaccumulation of heavy metals than the columella.

Toxicity of manganese to various life stages of selected marine cnidarian species.

Manganese (Mn) pollution in marine waters is increasing and sensitivities to this metal vary widely among marine species. The aims of this study were to characterise Mn chemistry in seawater, and evaluate the toxic effects of Mn on various life stages of two scleractinian corals - the branching sp. Acropora spathulata and massive sp. Platygyra daedalea, and the anemone Exaiptasia pallida. Analytical and theoretical characterisation experiments showed that 97-100% of Mn (II) additions ≤ 200 mg/L in seawater were soluble over 72 h and largely assumed labile complexes. Concentrations estimated to reduce coral fertilisation success by 50% (5.5-h EC50) were 237 mg/L for A. spathulata and 164 mg/L for P. daedalea. A relatively low 72-h LC50 of 7 mg/L was calculated for A. spathulata larvae. In a pilot test using fragments of adult A. spathulata, intact coral tissue rapidly sloughed away from the underlying skeleton at very low concentrations with a 48-h EC50 of just 0.7 mg/L. For E. pallida, survival, tentacle retraction and reproduction were unaffected by prolonged high exposures (12-d NOEC 54 mg/L). This study provides important data supporting the derivation of separate water quality guidelines for Mn in systems with and without coral - a decision recently considered by Australian and New Zealand authorities. It demonstrates the high sensitivity of coral larvae and adult colonies to Mn and the potential risks associated with relying on other early life stage tests and/or E. pallida as ecotoxicological representatives of critically important scleractinian corals.

Microplastics disturb the anthozoan-algae symbiotic relationship.

World production of plastic has dramatically increased from the 1950's and now it reaches approximately 311 million tons per year. The resulting accumulation of small plastic detritus less than 5 mm in size, termed "microplastics", has started threatening the life cycles of marine organisms. Here we show the first evidence that microplastics disturb the initiation of symbiotic relationships in anthozoan-algae symbiosis. We found in both the aposymbiotic sea-anemone Aiptasia sp. and the coral Favites chinensis that the infectivity of symbiotic algae into the host is severely suppressed by microspheres fed either directly or indirectly through microsphere-fed Artemia sp. Similar trends were seen when microplastics collected from commercial facewash were used instead of microspheres. Therefore, ongoing accumulation of microplastics in the ocean might disturb the healthy anthozoan-algae symbiotic relationships, which are cornerstones of the biologically enriched coral reef ecosystem.

Intraspecific variation in oxidative stress tolerance in a model cnidarian: Differences in peroxide sensitivity between and within populations of Nematostella vectensis.

Nematostella vectensis is a member of the phylum Cnidaria, a lineage that includes anemones, corals, hydras, and jellyfishes. This estuarine anemone is an excellent model system for investigating the evolution of stress tolerance because it is easy to collect in its natural habitat and to culture in the laboratory, and it has a sequenced genome. Additionally, there is evidence of local adaptation to environmental stress in different N. vectensis populations, and abundant protein-coding polymorphisms have been identified, including polymorphisms in proteins that are implicated in stress responses. N. vectensis can tolerate a wide range of environmental parameters, and has recently been shown to have substantial intraspecific variation in temperature preference. We investigated whether different clonal lines of anemones also exhibit differential tolerance to oxidative stress. N. vectensis populations are continually exposed to reactive oxygen species (ROS) generated during cellular metabolism and by other environmental factors. Fifteen clonal lines of N. vectensis collected from four different estuaries were exposed to hydrogen peroxide. Pronounced differences in survival and regeneration were apparent between clonal lines collected from Meadowlands, NJ, Baruch, SC, and Kingsport, NS, as well as among 12 clonal lines collected from a single Cape Cod marsh. To our knowledge, this is the first example of intraspecific variability in oxidative stress resistance in cnidarians or in any marine animal. As oxidative stress often accompanies heat stress in marine organisms, resistance to oxidative stress could strongly influence survival in warming oceans. For example, while elevated temperatures trigger bleaching in corals, oxidative stress is thought to be the proximal trigger of bleaching at the cellular level.

Modulation of activation and inactivation by Ca2+ and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis.

The archetypal TRPM2-like channel of the sea anemone Nematostella vectensis is gated by ADPR like its human orthologue but additionally exhibits properties of other vertebrate TRPM channels. Thus it can help towards an understanding of gating and regulation of the whole subfamily. To elucidate further the role of Ca2+ as a co-factor of ADPR, we exploited 2-aminoethyl diphenylborinate (2-APB), previously shown to exert either inhibitory or stimulatory effects on diverse TRPM channels, or both in a concentration-dependent manner. 2-APB in high concentrations (1 mM) induced large, non-inactivating currents through nvTRPM2. In lower concentrations (≤0.5 mM), it prevented the fast current inactivation typical for nvTRPM2 stimulated with ADPR. Both these effects were rapidly reversed after wash-out of 2-APB, in contrast to a considerable lag time of their onset. A detailed analysis of nvTRPM2 mutants with modified selectivity filter or reduced ADP-ribose sensitivity revealed that the actions of 2-APB depend on its access to the pore which is enhanced by channel opening. Moreover, access of Ca2+ to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca2+ on the pore can be discriminated with the aid of 2-APB.

Characterization of MXR activity in the sea anemone Bunodosoma cangicum exposed to copper.

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6µg/L) in presence and absence of MXR blocker (verapamil 50µM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6µg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8µgCu/L, but 15.6µgCu/L was similar to 0 and 7.8µg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves.

Toxicity estimates for diuron and atrazine for the tropical marine cnidarian Exaiptasia pallida and in-hospite Symbiodinium spp. using PAM chlorophyll-a fluorometry.

Effective ecotoxicological risk assessments for herbicides in tropical marine environments are restricted by a lack of toxicity data, sensitive test methods and endpoints for relevant species, and this requires rectification. The symbiotic sea anemone Exaiptasia pallida is a suitable test species, representing the phylum Cnidaria and allowing for assessments of toxicological responses of both the animal host and in-hospite Symbiodinium spp. Pulse amplitude modulated (PAM) chlorophyll-a fluorometry is recognised as a valuable ecotoxicological tool, and here newly-developed test methods are presented using PAM fluorometry to measure herbicide effects on photosynthetic efficiency of in-hospite Symbiodinium spp. Additionally, measurements on healthy laboratory-reared E. pallida provide baseline data demonstrating the normal effective quantum yield (EQY) and the maximum electron transport rate (ETRm) for Symbiodinium spp. in the absence of herbicide stress. Concentration-dependant reductions in the EQY and ETRm occurred during diuron and atrazine exposures; a mean 48-h EC50 (effective concentration; 50%) of 8µg/L of diuron was estimated, however atrazine elicited a much lower toxicity. Twelve-day exposures to 10-200µg/L diuron showed that the greatest EQY effect occurred during the first 48h, with little subsequent change. However, longer exposures to the lowest diuron treatment (1µg/L) showed the lowest EQYs after 96h followed by recovery to control levels within 12d. Furthermore, asexual reproduction was inhibited during 12-d exposures to diuron, and 12-d EC50 values of 100 and 132µg/L were estimated to inhibit successful reproduction of pedal lacerates and juveniles by 50% respectively. This study provides much needed data contributions to species sensitivity curves for development of diuron and atrazine water quality guidelines in tropical marine environments.

Assessing the chronic toxicity of copper and aluminium to the tropical sea anemone Exaiptasia pallida.

The world's most productive bauxite mines and alumina refineries are located in tropical or sub-tropical regions. The discharge water from alumina refineries can contain elevated aluminium (Al, <0.45µm fraction), from 30 to 1000µg/L. There is a need for additional information on the toxicity of Al to aquatic organisms to improve the environmental regulation and management of alumina refinery operations in tropical coastal regions. A 14-d chronic toxicity test was developed for the tropical sea anemone Exaiptasia pallida. Asexual reproduction and growth rates of E. pallida were assessed using the number of lacerates produced and oral disc diameter. The comparative sensitivity of E. pallida was assessed through exposure to a commonly-used reference toxicant, copper (Cu) at 28°C, with asexual reproduction toxicity estimates of 10% (EC10) and 50% (EC50) effect concentrations, calculated as 8.8µg/L (95% confidence limits (CL): 1-18µg/L) and 35µg/L Cu (95% CL: 30-39µg/L), respectively. Growth rate was a suitable additional endpoint (EC50=35µg/L Cu, 95% CL: 23-49µg/L). The EC10 and EC50 for Al (total fraction, based on reproduction) at 28°C were 817µg/L (95% CL: 440-1480µg/L) and 2270µg/L (95% CL: 1600-3900µg/L), respectively. The toxicity of Cu and Al was also assessed at 24°C and 31°C, representing average year-round water temperatures for sub-tropical and tropical Australian coastal environments. Changing the temperature from 28°C to 24°C or 31°C resulted in up to 45% less reproduction of anemones and increased their sensitivity to Cu (EC50s at 24°C=21µg/L, 95% CL: 17-26µg/L and at 31°C=23µg/L, 95% CL: 21-25µg/L). Sensitivity to Al was reduced at 24°C with an EC50 of 8870µg/L (95% CL: 6200-NC). An EC50 for Al at 31°C could not be calculated. This test is a reliable and sensitive addition to the suite of standardised tests currently developed for tropical marine species.

Responses of the sea anemone, Exaiptasia pallida, to ocean acidification conditions and zinc or nickel exposure.

Ocean acidification, caused by increasing atmospheric carbon dioxide (CO2), is a growing concern in marine environments. Land-based sources of pollution, such as metals, have also been a noted problem; however, little research has addressed the combined exposure of both pollutants to coral reef organisms. In this study we examined tissue metal accumulation and physiological effects (activity of anti-oxidant enzymes, catalase and glutathione reductase) in the sea anemone, Exaiptasia pallida after exposure to increased CO2, as well as zinc (Zn) or nickel (Ni). After exposure to four concentrations (nominal values=control, 10, 50, 100µg/L) of Zn or Ni over 7days, both metals accumulated in the tissues of E. pallida in a concentration-dependent manner. Anemones exposed to elevated CO2 (1000ppm) accumulated significant tissue burdens of Zn or Ni faster (by 48h) than those exposed to the same metal concentrations at ambient CO2. No differences were observed in catalase activity due to Zn exposure; however, 50µg/L Ni caused a significant increase in catalase activity at ambient CO2. No significant effect on catalase activity from CO2 exposure alone was observed. Glutathione reductase activity was affected by increased Zn or Ni exposure and those effects were influenced by increased CO2. Results of this study provide insight into the toxic mechanisms and environmental implications of CO2 and Zn or Ni exposure to the cnidarian E. pallida.

The stable microbiome of inter and sub-tidal anemone species under increasing pCO2.

Increasing levels of pCO2 within the oceans will select for resistant organisms such as anemones, which may thrive under ocean acidification conditions. However, increasing pCO2 may alter the bacterial community of marine organisms, significantly affecting the health status of the host. A pH gradient associated with a natural volcanic vent system within Levante Bay, Vulcano Island, Italy, was used to test the effects of ocean acidification on the bacterial community of two anemone species in situ, Anemonia viridis and Actinia equina using 16 S rDNA pyrosequencing. Results showed the bacterial community of the two anemone species differed significantly from each other primarily because of differences in the Gammaproteobacteria and Epsilonproteobacteria abundances. The bacterial communities did not differ within species among sites with decreasing pH except for A. viridis at the vent site (pH = 6.05). In addition to low pH, the vent site contains trace metals and sulfide that may have influenced the bacteria community of A. viridis. The stability of the bacterial community from pH 8.1 to pH 7.4, coupled with previous experiments showing the lack of, or beneficial changes within anemones living under low pH conditions indicates that A. viridis and A. equina will be winners under future ocean acidification scenarios.

In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis.

We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24 h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2 h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72 h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R 2 = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2 h after isolation and then replaced by standard medium, showed, at 6 days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

Responses of the sea anemone, Exaiptasia pallida, to ocean acidification conditions and copper exposure.

Ocean acidification (OA) is a growing concern due to its deleterious effects on aquatic organisms. Additionally, the combined effects of OA and other local stressors like metal pollution are largely unknown. In this study, we examined physiological effects in the sea anemone, Exaiptasia pallida after exposure to the global stressor carbon dioxide (CO2), as well as the local stressor copper (Cu) over 7 days. Cu accumulated in the tissues of E. pallida in a concentration-dependent manner. At some time points, sea anemones exposed to 1000 ppm CO2 had higher tissue Cu concentrations than those exposed to 400 ppm CO2 at the same Cu exposure concentrations. In general, the activities of all anti-oxidant enzymes measured (catalase, CAT; glutathione peroxidase, GPx, glutathione reductase, GR) increased with exposure to increasing Cu concentrations. Significant differences in GR, CAT and to some degree GPx activity, were observed due to increasing CO2 exposure in control treatments. Sea anemones exposed to Cu in combination with higher CO2 generally had higher anti-oxidant enzyme activities than those exposed to the same concentration of Cu and lower CO2. Activity of the enzyme, carbonic anhydrase (CA), involved in acid-base balance, was significantly decreased with increasing Cu exposure. At the two lowest Cu concentrations, the extent of CA inhibition was lessened with increasing CO2 concentration. These results provide insight into toxic mechanisms of both Cu and CO2 exposure to the sensitive cnidarian E. pallida and have implications for environmental exposure of multiple contaminants.

Nutrient enrichment coupled with sedimentation favors sea anemones over corals.

Fine sediments, which account for the majority of total fluvial sediment flux, have been suggested to degrade coral reefs on a global scale. Furthermore, sediment impacts can be exacerbated by extreme rainfall events associated with global climate change and anthropogenic nutrient enrichment. We report the findings from a series of mesocosm experiments exploring the effects of short-term sedimentation and nutrient enrichment on the interactions between the hard coral Acropora muricata, the sea anemone Mesactinia ganesis, and the green macroalga Codium edule. Mesocosms were manipulated to simulate either unimpacted reefs or reefs exposed to elevated levels of fine sediments for 10 or 14 days to simulate the effects of heavy rainfall. The first and second experiments were aimed to examine the effects of inorganic and organic sediments, respectively. The third experiment was designed to examine the interactive effects of nutrient enrichment and elevated sediment loads. Neither inorganic nor organic sediment loadings significantly affected the physiological performance of the coral, but, importantly, did reduce its ability to compete with other organisms. Photosynthetic efficiencies of both the green macroalga and the sea anemone increased in response to both sediment loadings when they were simultaneously exposed to nutrient enrichment. While organic sediment loading increased the nitrogen content of the green macroalga in the first experiment, inorganic sediment loading increased its phosphorus content in the second experiment. The coral mortality due to sea anemones attack was significantly greater upon exposure to enriched levels of organic sediments and nutrients. Our findings suggest that the combined effects of short-term sedimentation and nutrient enrichment could cause replacement of corals by sea anemones on certain coral reefs.

Comparative effects of dissolved copper and copper oxide nanoparticle exposure to the sea anemone, Exaiptasia pallida.

Increasing use of metal oxide nanoparticles (NP) by various industries has resulted in substantial output of these NP into aquatic systems. At elevated concentrations, NP may interact with and potentially affect aquatic organisms. Environmental implications of increased NP use are largely unknown, particularly in marine systems. This research investigated and compared the effects of copper oxide (CuO) NP and dissolved copper, as copper chloride (CuCl2), on the sea anemone, Exaiptasia pallida. Sea anemones were collected over 21 days and tissue copper accumulation and activities of the enzymes: catalase, glutathione peroxidase, glutathione reductase, and carbonic anhydrase were quantified. The size and shape of CuO NP were observed using a ecanning electron microscope (SEM) and the presence of copper was confirmed by using Oxford energy dispersive spectroscopy systems (EDS/EDX). E. pallida accumulated copper in their tissues in a concentration- and time-dependent manner, with the animals exposed to CuCl2 accumulating higher tissue copper burdens than those exposed to CuO NP. As a consequence of increased copper exposure, as CuO NP or CuCl2, anemones increased activities of all of the antioxidant enzymes measured to some degree, and decreased the activity of carbonic anhydrase. Anemones exposed to CuO NP generally had higher anti-oxidant enzyme activities than those exposed to the same concentrations of CuCl2. This study is useful in discerning differences between CuO NP and dissolved copper exposure and the findings have implications for exposure of aquatic organisms to NP in the environment.