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.

Collective Locomotion of Human Cells, Wound Healing and Their Control by Extracts and Isolated Compounds from Marine Invertebrates.

The collective migration of cells is a complex integrated process that represents a common theme joining morphogenesis, tissue regeneration, and tumor biology. It is known that a remarkable amount of secondary metabolites produced by aquatic invertebrates displays active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of crude extracts or isolated compounds that exert a modulatory effect on collective cell locomotion and/or skin tissue reconstitution and recapitulate the molecular, biochemical, and/or physiological aspects, where available, which are associated to the substances under examination, grouping the producing species according to their taxonomic hierarchy. Taken all of the collected data into account, marine invertebrates emerge as a still poorly-exploited valuable resource of natural products that may significantly improve the process of skin regeneration and restrain tumor cell migration, as documented by in vitro and in vivo studies. Therefore, the identification of the most promising invertebrate-derived extracts/molecules for the utilization as new targets for biomedical translation merits further and more detailed investigations.

Molecular assessment of wild populations in the marine realm: Importance of taxonomic, seasonal and habitat patterns in environmental monitoring.

Scientists are currently faced with the challenge of assessing the effects of anthropogenic stressors on aquatic ecosystems. Cellular stress response (CSR) biomarkers are ubiquitous and phylogenetically conserved among metazoans and have been successfully applied in environmental monitoring but they can also vary according to natural biotic and abiotic factors. The reported variability may thus limit the wide application of biomarkers in monitoring, imposing the need to identify variability levels in the field. Our aim was to carry out a comprehensive in situ assessment of the CSR (heat shock protein 70 kDa, ubiquitin, antioxidant enzymes) and oxidative damage (lipid peroxidation) in wild populations across marine taxa by collecting fish, crustaceans, mollusks and cnidarians during two different seasons (spring and summer) and two habitat types (coast and estuary). CSR end-point patterns were different between taxa with mollusks having higher biomarker levels, followed by the cnidarians, while fish and crustaceans showed lower biomarker levels. The PCA showed clear clusters related to mobility/sessile traits with sessile organisms showing greater levels (>2-fold) of CSR proteins and oxidative damage. Mean intraspecific variability in the CSR measured by the coefficient of variation (% CV) (including data from all seasons and sites) was elevated (35-94%). Overall, there was a seasonal differentiation in biomarker patterns across taxonomic groups, especially evident in fish and cnidarians. A differentiation in biomarker patterns between habitat types was also observed and associated with phenotypic plasticity or local adaptation. Overall, specimens collected in the estuary had lower biomarker levels when compared to specimens collected in the coast. This work highlights the importance of assessing baseline biomarker levels across taxa, seasons and habitats prior to applying biomarker analyses in environmental monitoring. Selecting bioindicator species, defining sampling strategies, and identifying confounding factors are crucial preliminary steps that ensure the success of biomarkers as powerful tools in biomonitoring.

Comparison of the Inducing Effect of Indole Compounds on Medusa Formation in Different Classes of Medusozoa.

Scyphozoa, Cubozoa and Hydrozoa are classes in the phylum Cnidaria that undergo metagenesis involving a dramatic morphological transition. In Scyphozoa and Cubozoa, when exposed to species- or strain-specific transition-inducing stimuli, asexually reproducing benthic polyps transform into sexually reproducing planktonic medusae. In Hydrozoa, exposure to species- or strainspecific transition-inducing stimuli causes formation of medusa buds in the polyp's body. In Aurelia aurita (Linnaeus, 1758) (Scyphozoa, Semaeostomeae), polyp-to-jellyfish transition is induced by some simple indole compounds. However, whether indole compounds can induce polyp-to-jellyfish transition in Cubozoa and Hydrozoa remains unknown. In the present study, we show that an indole compound, 5-methoxy-2-methylindole, induces polyp-to-jellyfish transition in Scyphozoa and Cubozoa. This inducing action suggests that the downstream steps of polyp-to-jellyfish transition are regulated by the same biochemical reactions in Scyphozoa and Cubozoa, irrespective of the type of transition-inducing environmental stimuli.

MAPK signaling is necessary for neurogenesis in Nematostella vectensis.

BACKGROUND: The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood. RESULTS: We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages. CONCLUSIONS: Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.

Comparative sensitivity of the cnidarian Exaiptasia pallida and a standard toxicity test suite: testing whole effluents intended for ocean disposal.

The sea anemone Exaiptasia pallida (formally Aiptasia pulchella) has been identified as a valuable test species for tropical marine ecotoxicology. Here, the sensitivities of newly developed endpoints for E. pallida to two unidentified whole effluents were compared to a standard suite of temperate toxicity test species and endpoints that are commonly used in toxicological risk assessments for tropical marine environments. For whole effluent 1 (WE1), a 96-h lethal concentration 50 % (LC50) of 40 (95 % confidence intervals, 30-54) % v/v and a 12-day LC50 of 12 (9-15) % v/v were estimated for E. pallida, exhibiting a significantly higher sensitivity than standard sub-lethal endpoints in Allorchestes compressa (96-h effective concentration 50 % (EC50) of >100 % v/v for immobilisation) and Hormosira banksii (72-h EC50 of >100 % v/v for germination), and a similar sensitivity to Mytilus edulis galloprovincialis larval development with a 48-h LC50 of 29 (28-30) % v/v. Sub-lethal effects of whole effluent 2 (WE2) on E. pallida pedal lacerate development resulted in an 8-day EC50 of 7 (3-11) % v/v, demonstrating comparable sensitivity of this endpoint to standardised sub-lethal endpoints in H. banksii (72-h EC50 of 11 (10-11) % v/v for germination), M. edulis galloprovincialis (48-h EC50 for larval development of 12 (9-14) % v/v) and Heliocidaris tuberculata (1-h EC50 of 13 (12-14) % v/v for fertilisation; 72-h EC50 of 26 (25-27) % v/v for larval development) and a significantly higher sensitivity than A. compressa immobilisation (96-h EC50 of >100 % v/v). The sensitivity of E. pallida compared to a standard test species suite highlights the value in standardising the newly developed toxicity test methods for inclusion in routine toxicological risk assessment of complex whole effluents. Importantly, this species provides an additional taxonomic group to the test species that are currently available for tropical marine ecotoxicology and, being a cnidarian, may represent important tropical marine environments including coral reefs.

Heavy metals affect nematocysts discharge response and biological activity of crude venom in the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa).

BACKGROUND: Pollution of marine ecosystems and, specifically, heavy metals contamination may compromise the physiology of marine animals with events occurring on a cellular and molecular level. The present study focuses on the effect of short-term exposure to heavy metals like Zinc, Cadmium, Cobalt and Lanthanum (2-10 mM) on the homeostasis of Pelagia noctiluca (Cnidaria, Scyphozoa), a jellyfish abundant in the Mediterranean sea. This species possesses stinging organoids, termed nematocysts, whose discharge and concomitant delivery of venom underlie the survival of all Cnidaria. METHODS: Nematocysts discharge response, elicited by combined chemico-physical stimulation, was verified on excised oral arms exposed to heavy metals for 20 min. In addition, the hemolytic activity of toxins, contained in the crude venom extracted from nematocysts isolated from oral arms, was tested on human erythrocytes, in the presence of heavy metals or their mixture. RESULTS: Treatment with heavy metals significantly inhibited both nematocysts discharge response and hemolytic activity of crude venom, in a dose-dependent manner, not involving oxidative events, that was irreversible in the case of Lanthanum. CONCLUSION: Our findings show that the homeostasis of Pelagia noctiluca, in terms of nematocysts discharge capability and effectiveness of venom toxins, is dramatically and rapidly compromised by heavy metals and confirm that this jellyfish is eligible as a model for ecotoxicological investigations.

Nematocyst discharge in Pelagia noctiluca (Cnidaria, Scyphozoa) oral arms can be affected by lidocaine, ethanol, ammonia and acetic acid.

Nematocyst discharge and concomitant delivery of toxins is triggered to perform both defence and predation strategies in Cnidarians, and may lead to serious local and systemic reactions in humans. Pelagia noctiluca (Cnidaria, Scyphozoa) is a jellyfish particularly abundant in the Strait of Messina (Italy). After accidental contact with this jellyfish, not discharged nematocysts or even fragments of tentacles or oral arms may tightly adhere to the human skin and, following discharge, severely increase pain and the other adverse consequences of the sting. The aim of the present study is to verify if the local anesthetic lidocaine and other compounds, like alcohols, acetic acid and ammonia, known to provide pain relief after jellyfish stings, may also affect in situ discharge of nematocysts. Discharge was induced by a combined physico-chemical stimulation of oral arms by chemosensitizers (such as N-acetylated sugars, aminoacids, proteins and nucleotides), in the presence or absence of 1% lidocaine, 70% ethanol, 5% acetic acid or 20% ammonia, followed by mechanical stimulation by a non-vibrating test probe. The above mentioned compounds failed to induce discharge per se, and dramatically impaired the chemosensitizer-induced discharge response. We therefore suggest that prompt local treatment of the stung epidermis with lidocaine, acetic acid, ethanol and ammonia may provide substantial pain relief and help in reducing possible harmful local and systemic adverse reaction following accidental contact with P. noctiluca specimens.

Stress and death of cnidarian host cells play a role in cnidarian bleaching.

Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis.

Heavy metals affect regulatory volume decrease (RVD) in nematocytes isolated from the jellyfish Pelagia noctiluca.

The environmental contamination caused by heavy metals raises the question of their effect on biological systems. Among bio-indicators useful to monitor the toxicological effects of these chemicals, Cnidarians offer a unique model. Cnidarians possess highly specialized stinging cells, termed nematocytes, which respond to hyposmotic solution with well established homeostatic parameters as an acute osmotic phase (OP), leading to cell swelling, and then a slower regulatory volume decrease (RVD) phase, causing cell shrinkage. Here we report the effect of 65% artificial sea water (ASW) containing heavy metals, such as Cd, La, Co, Cu and Zn (concentrations comprised between 100 and 0.1 µM) on both OP and RVD in nematocytes isolated from the jellyfish Pelagia noctiluca by 605 mM NaSCN plus 0.01 mM Ca(2+). The exposure of the cells to Co and La inhibited RVD but not OP. However, Cu, Cd and Zn prevented the OP in a dose-dependent manner and, hence, also the detection of RVD. These results suggest that, in isolated nematocytes, heavy metal pollutants impair RVD either directly or indirectly through interference with the OP, thus negating RVD. Although further studies need to clarify the exact mechanisms whereby heavy metals exert their toxicity, it is evident that nematocytes of Cnidarians could serve as a model for ecotoxicological investigations.

Methodological approaches for nanotoxicology using cnidarian models.

The remarkable amenability of aquatic invertebrates to laboratory manipulation has already made a few species belonging to the phylum Cnidaria as attracting systems for exploring animal development. The proliferation of molecular and genomic tools, including the whole genomic sequence of the freshwater polyp Hydra vulgaris and the starlet sea anemone Nematostella vectensis, further enhances the promise of these species to investigate the evolution of key aspects of development biology. In addition, the facility with which cnidarian population can be investigated within their natural ecological context suggests that these models may be profitably expanded to address important questions in ecology and toxicology. In this review, we explore the traits that make Hydra and Nematostella exceptionally attractive model organisms in context of nanotoxicology, and highlight a number of methods and developments likely to further increase that utility in the near future.

Allorecognition triggers autophagy and subsequent necrosis in the cnidarian Hydractinia symbiolongicarpus.

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

Symbiodinium isolation by NaOH treatment.

The presence of photosynthetic zooxanthellae (dinoflagellates) in the tissue of many cnidarians is the main reason for their ecological success (i.e. coral reefs). It could also be the main cause of their demise, as the worldwide bleaching of reef-building coral is nothing less than the breakdown of this symbiotic association. The stability of this relationship is the principal marker for the biomonitoring of cnidarian health. We have therefore developed a new, simple method to isolate zooxanthellae in a few steps using NaOH solution. The protocol was validated in three symbiotic cnidarian species: a sea anemone, a gorgonian and a coral. Our method allows the isolation of intact and viable zooxanthellae with better yields than classic methods, especially for species with a calcareous skeleton. Moreover, the isolated zooxanthellae were free of host nucleic contaminants, facilitating subsequent specific molecular analyses.

Effect of halogenated fluorescent compounds on bioluminescent reactions.

The paper investigates an application of luminescent bioassays to monitor the toxicity of organic halides. Effects of xanthene dyes (fluorescein, eosin Y, and erythrosin B), used as model compounds, on bioluminescent reactions of firefly Luciola mingrelica, marine bacteria Photobacterium leiognathi, and hydroid polyp Obelia longissima were studied. Dependence of bioluminescence quenching constants on the atomic weight of halogen substituents in dye molecules was demonstrated. Bacterial bioluminescence was shown to be most sensitive to heavy halogen atoms involved in molecular structure; hence, it is suitable for construction of sensors to monitor toxicity of halogenated compounds. Mechanisms of bioluminescence quenching--energy transfer processes, collisional interactions, and enzyme-dye binding--were considered. Changes of bioluminescence (BL) spectra in the presence of the dyes were analyzed. Interactions of the dyes with enzymes were studied using fluorescence characteristics of the dyes in steady-state and time-resolved experiments. The dependences of fluorescence anisotropy of enzyme-bound dyes, the average fluorescence lifetime, and the number of exponential components in fluorescence decay on the atomic weight of halogen substituents were demonstrated. The results are discussed in terms of "dark effect of heavy halogen atom" in the process of enzyme-dye binding; hydrophobic interactions were assumed to be responsible for the effect.

Ecotoxicity of a brominated flame retardant (tetrabromobisphenol A) and its derivatives to aquatic organisms.

The large use of tetrabromobisphenol A (B(4)BPA) in common products (plastics, electric and electronic equipments) has raised concern about its ecotoxicity. Physical and bio-degradations may lead to the formation of tetrabromobisphenol A derivatives like tri- (B(3)BPA), di- (B(2)BPA), monobromobisphenol A (B(1)BPA) and bisphenol A (BPA). However, little is known about the toxicity of these brominated derivatives. An appraisal on the ecotoxicity of B(4)BPA and its derivatives was carried out with several bioassays representing organisms (bacteria, algae, micro-invertebrates and fish) of different taxonomic groups present in aquatic ecosystems. Endpoint values showed that B(4)BPA was significantly less toxic than the other chemicals when tested with the Microtox and algal asssays. A similar trend was observed with other bioassays for BPA. One of the brominated derivatives was particularly toxic: B(2)BPA. The LuminoTox assay and the rainbow trout hepatocytes assay reported the most significant toxicity for this derivative. Its toxicity was also significantly higher than the other compounds barring B(3)BPA when tested with the micro-crustacean test.

Sub-cellular damage by copper in the cnidarian Zoanthus robustus.

Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110 microg Cu L(-1)). A decrease in net photosynthesis was observed only at the highest copper concentration (156 microg Cu L(-1)). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae.

Effects of freezing, drying, ultraviolet irradiation, chlorine, and quaternary ammonium treatments on the infectivity of myxospores of Myxobolus cerebralis for Tubifex tubifex.

The effects of freezing, drying, ultraviolet irradiation (UV), chlorine, and a quaternary ammonium compound on the infectivity of the myxospore stage of Myxobolus cerebralis (the causative agent of whirling disease) for Tubifex tubifex were examined in a series of laboratory trials. Freezing at either -20 degrees C or -80 degrees C for a period of 7 d or 2 months eliminated infectivity as assessed by the absence of production of the actinospore stage (triactinomyxons [TAMs]) from T. tubifex cultures inoculated with treated myxospores over a 4-5-month period. Myxospores retained infectivity when held in well water at 5 degrees C or 22 degrees C for 7 d and when held at 4 degrees C or 10 degrees C d for 2 months. In contrast, no TAMs were produced from T. tubifex cultures inoculated with myxospores held at 20 degrees C for 2 months. Drying of myxospores eliminated any evidence of infectivity for T. tubifex. Doses of UV from 40 to 480 mJ/cm2 were all effective for inactivating myxospores of M. cerebralis, although a few TAMs were detected in one replicate T. tubifex culture at 240 mJ/cm2 and in one replicate culture at 480 mJ/cm2. Treatments of myxospores with chlorine bleach at active concentrations of at least 500 mg/L for 15 min largely inactivated myxospore infectivity for T. tubifex. Likewise, there was no evidence of TAMs produced by T. tubifex inoculated with myxospores treated with alkyl dimethyl benzyl ammonium chloride (ADBAC) at 1,500 mg/L for 10 min. Treatments of myxospores with 1,000-mg/L ADBAC for 10 min reduced TAM production in T. tubifex cultures sevenfold relative to that in cultures inoculated with an equal number of untreated myxospores. These results indicate that myxospores of M. cerebralis demonstrate a selective rather than broad resistance to selected physical and chemical treatments, and this selective resistance is consistent with conditions that myxospores are likely to experience in nature.

Biochemical responses of cnidarian larvae to mercury and benzo(a)pyrene exposure.

The biochemical responses of planulae from the coral Porites astreoides exposed to 10 microg/L of benzo(a)pyrene (B(a)P) and to 10 microg/L of mercury (Hg) was evaluated. The survivorship of larvae only dropped significantly after 48 h of B(a)P exposure, whereas it remained at 98% for Hg exposure and up to 96 h. Exposure to B(a)P significantly increased free thiols, and the activity of glutathione-S-transferase and catalase were unaltered under exposure of any of the contaminants. This study is the first contribution of the biochemical effects in cnidarian larvae exposed to contaminants.

Differential effects of tributyltin and copper antifoulants on recruitment of non-indigenous species.

Maritime transport is a primary vector for many marine invaders. For the past two decades, most commercial vessels have used tributyltin (TBT) antifouling (AF) paint, whereas recreational vessels have been restricted to alternatives, most commonly containing copper. Settlement plates painted with a collar of copper or TBT AF paint, and unpainted control plates, were deployed in commercial and recreational embayments in Port Jackson, Australia, and sampled photographically after 5 and 10 months. Copper enhanced early recruitment of several non-indigenous species (NIS), whereas recruitment of indigenous species was typically reduced by copper. TBT limited the recruitment of NIS for just 5 months and indigenous species, for the entire study. The results suggest that the use of toxic AF paints, and the possible accumulation of AF biocides in embayments, may be negatively affecting indigenous epibiota. Conversely, copper antifoulants on recreational vessels may be facilitating the transport and establishment of copper tolerant NIS into disturbed estuarine habitats.

Reevaluation of ENCORE: support for the eutrophication threshold model for coral reefs.

The results from the multimillion dollar Enrichment of Nutrients on Coral Reefs Experiment (ENCORE) on One Tree Island Reef (OTIR) suggest that increased nutrient loads to coral reefs will have little or no effect on the algal growth rates and, hence, on the associated effects that increased algal growth might have on the functioning and stability of coral reefs. However, a comparison of the concentrations of nutrients within the OTIR lagoon with the proposed nutrient threshold concentrations (NTC) for coral reefs suggests that all sites, including the control sites, were saturated with nutrients during ENCORE, and, hence, one would not expect to get any differences between treatments in the algal-growth related measurements. Thus, ENCORE results provide strong support for the proposed NTCs and support the ecological principle that algal productivity and, consequently, the functioning of coral reefs are sensitive to small changes in the background concentrations of nutrients. The principal conclusion of ENCORE, namely that the addition of nutrients did not cause the "pristine" OTIR to convert from coral communities to algal dominated reefs, is contrary to the fact that there was prolific macroalgal growth on the walls and crests of the experimental microatolls by the end of ENCORE.