OBJECTIVE: This study aimed to present a case of transient corneal damage after exposure to the effluent squirting from a sea anemone, Anthopleura uchidai, and to experimentally confirm the presence of toxic substances from an A. uchidai in the tissue culture.
METHODS: We reviewed the clinical course of a 51-year-old man who complained of decreased vision in his left eye after the stinging of a sea anemone, A. uchidai. The toxicity of the effluents from an A. uchidai in immortalized human corneal endothelial cells (HCEnC-21T) and human corneal epithelial cells in vitro were evaluated.
RESULTS: Corneal edema was observed, and his best-corrected visual acuity was 0.2. Corneal endothelial cell density decreased to 1435 cells/mm2. Although his corneal edema and visual acuity recovered after topical instillation with a topical steroid and 5% NaCl, corneal endothelial cell density did not recover for 3 years after the injury. The in vitro study revealed fractioned effluence from the sea anemone, by size-exclusion chromatography, containing a substance toxic to HCEnC-21T with cytoplasmic swelling and nuclear dislocation.
CONCLUSIONS: It is necessary to be cautious of effluents from sea anemones along the coast, and ophthalmologists should be aware that sea anemones can cause corneal endothelial dysfunction.

Anemonia viridis is an abundant and widely distributed temperate sea anemone that can form dense congregations of individuals. Despite the potential severity of its sting, few detailed cases have been reported. We report a case of a severe toxic reaction following an A. viridis sting in a 35-year-old oceanographer. She developed severe pain, itching, redness, and burning sensation, which worsened one week after treatment with anti-inflammatories, antihistamines and corticosteroids. Prompted by this event, and due to the insufficient risk prevention, lack of training for marine-environment users, and lack of research into sting-specific first-aid protocols, we evaluated the cnidocyst response to five different compounds commonly recommended as rinse solutions in first-aid protocols (seawater, vinegar, ammonia, baking soda, and freshwater) by means of the Tentacle Solution Assay. Vinegar and ammonia triggered an immediate and massive cnidocyst discharge after their application and were classified as activator solutions. Baking soda and freshwater were also classified as activator solutions, although with a lower intensity of discharge. Only seawater was classified as a neutral solution and therefore recommended as a rinse solution after A. viridis sting, at least until an inhibitory solution is discovered.

Protein-lipid interactions are crucial events from a biochemical point of view, like the interaction of proteins with the cell plasma membrane, and their study is of great importance. Actinoporins are a very powerful tool to study this kind of interactions, since they are soluble proteins in an aqueous environment, capable of inserting into membranes when they have the adequate composition. In fact, actinoporins have been used to study protein-lipid interactions for many years now. Sometimes it is not possible to use real biological membranes in the experiments, so model membranes need to be used. This article aims to give a thorough description of many of the techniques used to study actinoporin-lipid interactions, using both biological and model membranes: Hemolysis, release of vesicles content, surface plasmon resonance, isothermal titration calorimetry, fluorescence-based measurements, etc. Some of these techniques measure the actinoporins activity and some measure their binding properties. The combination of all the techniques described can offer valuable information about the thermodynamics and the kinetics of the actinoporin-lipid interaction.

Animal regeneration is a biological process leading to the reformation of injured or lost tissues/body parts. One of the most fascinating regenerative phenomena is the so-called whole-body regeneration, leading to the reformation of fully functional organisms within days after bisection. The sea anemone Nematostella vectensis is currently emerging as novel whole-body regeneration model. Here we describe the methods of inducing the regenerative process in this cnidarian as well as the fixation and staining protocols for morphological, molecular, and cellular analysis.

BACKGROUND: Nervous systems originated before the split of Proto- and Deuterostomia, more than 600 million years ago. Four animal phyla (Cnidaria, Placozoa, Ctenophora, Porifera) diverged before this split and studying these phyla could give us important information on the evolution of the nervous system. Here, we have annotated the neuropeptide preprohormone genes of twenty species belonging to the subclass Hexacorallia or Ceriantharia (Anthozoa: Cnidaria), using thirty-seven publicly accessible genome or transcriptome databases. Studying hexacorals is important, because they are versatile laboratory models for development (e.g., Nematostella vectensis) and symbiosis (e.g., Exaiptasia diaphana) and also are prominent reef-builders.
RESULTS: We found that each hexacoral or ceriantharian species contains five to ten neuropeptide preprohormone genes. Many of these preprohormones contain multiple copies of immature neuropeptides, which can be up to 50 copies of identical or similar neuropeptide sequences. We also discovered preprohormones that only contained one neuropeptide sequence positioned directly after the signal sequence. Examples of them are neuropeptides that terminate with the sequence RWamide (the Antho-RWamides). Most neuropeptide sequences are N-terminally protected by pyroglutamyl (pQ) or one or more prolyl residues, while they are C-terminally protected by an amide group. Previously, we isolated and sequenced small neuropeptides from hexacorals that were N-terminally protected by an unusual L-3-phenyllactyl group. In our current analysis, we found that these N-phenyllactyl-peptides are derived from N-phenylalanyl-peptides located directly after the signal sequence of the preprohormone. The N-phenyllactyl- peptides appear to be confined to the hexacorallian order Actiniaria and do not occur in other cnidarians. On the other hand, (1) the neuropeptide Antho-RFamide (pQGRFamide); (2) peptides with the C-terminal sequence GLWamide; and (3) tetrapeptides with the X1PRX2amide consensus sequence (most frequently GPRGamide) are ubiquitous in Hexacorallia.
CONCLUSIONS: We found GRFamide, GLWamide, and X1PRX2amide peptides in all tested Hexacorallia. Previously, we discovered these three neuropeptide classes also in Cubozoa, Scyphozoa, and Staurozoa, indicating that these neuropeptides originated in the common cnidarian ancestor and are evolutionarily ancient. In addition to these ubiquitous neuropeptides, other neuropeptides appear to be confined to specific cnidarian orders or subclasses.

Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades. However, regardless of the probe of choice, the results reported that StnII action was never able to empty the vesicles completely. This was hard to explain if StnII pores were to be stable and always leaky for the probes used. To address this question, we have used a variety of probes, including rhodamine 6G or Tb3+, to test the permeability of StnII\'s pores. Our results indicate that calcein was in fact too large to fit through StnII\'s pores, and that the standard method in the field is actually reporting StnII-induced transient permeation of the membrane rather than the passage of solutes through the stable assembled pores. In order to evaluate the permeability of these structures, we used a dithionite-based assay, which showed that the final pores were in fact open. Thus, our results indicate that the stable actinoporins\' pores are open in spite of plateaued classic release curves. Besides the proper pore, the first stages of pore formation would inflict serious damage to living cells as well.

Coral reefs are faced with almost complete destruction by the end of the century due to global warming unless humanity can cap global temperature rise. There is now a race to develop a diverse set of solutions to save coral reefs. In this perspective, a case is made for understanding the cell biology of coral-dinoflagellate symbiosis to help inform development of solutions for saving reefs. Laboratory model systems for the study of coral symbiosis, including the sea anemone Exaiptasia pallida, are featured as valuable tools in the fight to save corals. The roles of host innate immunity and inter-partner nutrient dynamics in the onset, ongoing maintenance, and dysregulation of symbiosis are reviewed and discussed. Key innate immune genes and pathways, such as glycan-lectin interactions, the sphingosine rheostat, and the cytokine transforming growth factor beta are shown to modulate a host immune response in the symbiotic state. An upset in the homeostatic inorganic nutrient balance during heat stress and high exogenous nutrient availability is credited with driving the partnership toward dysregulation and coral bleaching. Specific examples are given where knowledge of the cell biology of symbiosis is informing the development of solutions, including studies showing clear limitations in the value of partner switching and acclimatization protocols. Finally, emphasis is placed on rapid advancement of knowledge to try to meet the urgent need for solutions. This includes real-time open communication with colleagues on successes and failures, sharing of resources and information, and working together in the spirit of a collective mission to save coral reefs.

The cnidarian Nematostella vectensis has become an established lab model, providing unique opportunities for venom evolution research. The Nematostella venom system is multimodal: involving both nematocytes and ectodermal gland cells, which produce a toxin mixture whose composition changes throughout the life cycle. Additionally, their modes of interaction with predators and prey vary between eggs, larvae, and adults, which is likely shaped by the dynamics of the venom system. Nv1 is a major component of adult venom, with activity against arthropods (through specific inhibition of sodium channel inactivation) and fish. Nv1 is encoded by a cluster of at least 12 nearly identical genes that were proposed to be undergoing concerted evolution. Surprisingly, we found that Nematostella venom includes several Nv1 paralogs escaping a pattern of general concerted evolution, despite belonging to the Nv1-like family. Here, we show two of these new toxins, Nv4 and Nv5, are lethal for zebrafish larvae but harmless to arthropods, unlike Nv1. Furthermore, unlike Nv1, the newly identified toxins are expressed in early life stages. Using transgenesis and immunostaining, we demonstrate that Nv4 and Nv5 are localized to ectodermal gland cells in larvae. The evolution of Nv4 and Nv5 can be described either as neofunctionalization or as subfunctionalization. Additionally, the Nv1-like family includes several pseudogenes being an example of nonfunctionalization and venom evolution through birth-and-death mechanism. Our findings reveal the evolutionary history for a toxin radiation and point toward the ecological function of the novel toxins constituting a complex cnidarian venom.

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

Studying the spatial gene expression profiles from in situ hybridization images of the embryo is one of the first steps toward the comprehensive understanding of gene interactions in an organism. In the case of N. vectensis, extracting and collecting these data is a challenging task due to the difficulty of detecting the cell layer through the transparent body plan and changing morphology during the blastula and gastrula stages. Here, first, we introduce a method to algorithmically identify and track the cell layer in N. vectensis embryo from the late blastula to the late gastrula stage. With this, we will be able to extract spatial expression profiles of genes alongside the cell layer and consequently reconstructing the 1D representation of gene expression profiles. Furthermore, we use the morphological configurations of the embryo extracted from confocal images, to model the dynamics of embryos morphology during the gastrulation process in 2D. Ultimately, we provide a visualization tool for studying and comparing the extracted spatial gene expression profiles over the simulated embryo. We anticipate that our method of extraction and visualization to be a starting point for quantifying and collecting more in situ images from various sources, which can potentially accelerate our understanding of gene interactions in the early development of N. vectensis. The method allows researchers to visualize and compare the different gene expressions from different in situ images or different experiments. As an example, we were able to show the complementary expression of NvFoxA-NvSnailA and NvBra-NvErg in the central domain and central/external rings during the development which suggests the possible repression effects between each pair; as it has been discovered by functional analysis.