Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis.

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.

The evolution of cnidarian stinging cells supports a Precambrian radiation of animal predators.

Cnidarians-the phylum including sea anemones, corals, jellyfish, and hydroids-are one of the oldest groups of predatory animals. Nearly all cnidarians are carnivores that use stinging cells called cnidocytes to ensnare and/or envenom their prey. However, there is considerable diversity in cnidocyte form and function. Tracing the evolutionary history of cnidocytes may therefore provide a proxy for early animal feeding strategies. In this study, we generated a time-calibrated molecular clock of cnidarians and performed ancestral state reconstruction on 12 cnidocyte types to test the hypothesis that the original cnidocyte was involved in prey capture. We conclude that the first cnidarians had only the simplest and least specialized cnidocyte type (the isorhiza) which was just as likely to be used for adhesion and/or defense as the capture of prey. A rapid diversification of specialized cnidocytes occurred through the Ediacaran (~654-574 million years ago), with major subgroups developing unique sets of cnidocytes to match their distinct feeding styles. These results are robust to changes in the molecular clock model, and are consistent with growing evidence for an Ediacaran diversification of animals. Our work also provides insight into the evolution of this complex cell type, suggesting that convergence of forms is rare, with the mastigophore being an interesting counterexample.

An ancient pan-cnidarian microRNA regulates stinging capsule biogenesis in Nematostella vectensis.

An ancient evolutionary innovation of a novel cell type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians to paralyze their prey and predators, contributing to this phylum's evolutionary success. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells in the sea anemone Nematostella vectensis. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis in Nematostella, while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we revealed a functional basis to the conservation of one of nature's most ancient microRNAs.

Effect of Rinse Solutions on Rhizostoma pulmo (Cnidaria: Scyphozoa) Stings and the Ineffective Role of Vinegar in Scyphozoan Jellyfish Species.

Rhizostoma pulmo is a widely distributed scyphozoan in the Mediterranean Sea. Their stings result mainly in erythema, small vesicles, or/and pain, and cause a high number of bathers to seek assistance from first-aid services during the summer season. Despite the threat that jellyfish stings represent to public health, there is disagreement in the scientific community on first-aid protocols, with the dispute largely centered around the effectiveness of vinegar. In the present research, we investigated the effect of commonly used rinse solutions on nematocyst discharge in R. pulmo and the effect of vinegar on three more scyphozoans (Aurelia sp., Cassiopea sp., and Rhizostoma luteum). Scented ammonia, vinegar, and acetic acid triggered nematocyst discharge in R. pulmo. Vinegar also caused nematocyst discharge in Aurelia sp., Cassiopea sp., and R. luteum. In contrast, seawater, baking soda, freshwater, urine, and hydrogen peroxide were considered neutral solutions that did not induce nematocyst discharge. These results indicate that the use of vinegar, acetic acid, or commercial products based on these compounds is counterproductive. Their use can worsen pain and discomfort caused not only by R. pulmo stings but also by those of any scyphozoan. The use of seawater is recommended for cleaning the R. pulmo sting site until an inhibitor solution that irreversibly prevents nematocyst discharge is discovered.

Inhibition of Nematocyst Discharge from Pelagia noctiluca (Cnidaria: Scyphozoa)-Prevention Measures against Jellyfish Stings.

Pelagia noctiluca stings are common in Mediterranean coastal areas and, although the venom is non-lethal, they are painful. Due to its high toxicity and abundance, P. noctiluca is considered a target species for the focus of research on active ingredients to reduce the symptoms of its sting. To determine the effect of 31 substances and formulations on nematocyst discharge, we performed three tests: (1) screening of per se discharge activator solutions, (2) inhibitory test with nematocyst chemical stimulation (5% acetic acid) and (3) inhibitory test quantifying the hemolytic area. Ammonia, barium chloride, bleach, scented ammonia, carbonated cola, lemon juice, sodium chloride and papain triggered nematocyst discharge. All of them were ruled out as potential inhibitors. Butylene glycol showed a reduction in nematocyst discharge, while the formulations of 10% lidocaine in ethanol, 1.5% hydroxyacetophenone in distilled water + butylene glycol, and 3% Symsitive® in butylene glycol inhibited nematocyst discharge. These last results were subsequently correlated with a significant decrease in hemolytic area in the venom assays versus seawater, a neutral solution. The presented data represent a first step in research to develop preventive products for jellyfish stings while at the same time attempting to clarify some uncertainties about the role of various topical solutions in P. noctiluca first-aid protocols.

Trial Assay for Safe First-Aid Protocol for the Stinging Sea Anemone Anemonia viridis (Cnidaria: Anthozoa) and a Severe Toxic Reaction.

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.

Magnesium concentration influences size and pulse rate in the upside-down jellyfish, Cassiopea andromeda.

Magnesium is involved in a variety of physiological processes in marine animals and is known to be deleterious in both excess and deficiency. The effects of magnesium concentration ranging from 700 mg/L (low), 1344 mg/L (control), and 2000 mg/L (high) on size and pulse rate in upside-down jellyfish (Cassiopea andromeda) medusae were examined in two separate 28-day trials. Exposure to low magnesium resulted in significantly (p < .05) higher pulse rates and decreased bell diameter and also produced oral arm degradation. Exposure to high magnesium resulted in significantly (p < .05) lower pulse rates and decreased bell diameter as well as oral arm cupping. In both low and high magnesium, almost all specimens changed color from pale blue on Day 1, to brown by Day 28, suggesting a loss of zooxanthellae. The decrease in bell diameter and color change was more pronounced and occurred more rapidly in low magnesium. The results of both trials demonstrate the deleterious effects of high and low magnesium on C. andromeda and emphasize the importance of monitoring magnesium concentration to maintain healthy display animals in public aquaria.

Cell type-specific expression profiling unravels the development and evolution of stinging cells in sea anemone.

BACKGROUND: Cnidocytes are specialized cells that define the phylum Cnidaria. They possess an "explosive" organelle called cnidocyst that is important for prey capture and anti-predator defense. An extraordinary morphological and functional complexity of the cnidocysts has inspired numerous studies to investigate their structure and development. However, the transcriptomes of the cells bearing these unique organelles are yet to be characterized, impeding our understanding of the genetic basis of their biogenesis. RESULTS: In this study, we generated a nematocyte reporter transgenic line of the sea anemone Nematostella vectensis using the CRISPR/Cas9 system. By using a fluorescence-activated cell sorter (FACS), we have characterized cell type-specific transcriptomic profiles of various stages of cnidocyte maturation and showed that nematogenesis (the formation of functional cnidocysts) is underpinned by dramatic shifts in the spatiotemporal gene expression. Among the genes identified as upregulated in cnidocytes were Cnido-Jun and Cnido-Fos1-cnidarian-specific paralogs of the highly conserved c-Jun and c-Fos proteins of the stress-induced AP-1 transcriptional complex. The knockdown of the cnidocyte-specific c-Jun homolog by microinjection of morpholino antisense oligomer results in disruption of normal nematogenesis. CONCLUSIONS: Here, we show that the majority of upregulated genes and enriched biochemical pathways specific to cnidocytes are uncharacterized, emphasizing the need for further functional research on nematogenesis. The recruitment of the metazoan stress-related transcription factor c-Fos/c-Jun complex into nematogenesis highlights the evolutionary ingenuity and novelty associated with the formation of these highly complex, enigmatic, and phyletically unique organelles. Thus, we provide novel insights into the biology, development, and evolution of cnidocytes.

Evidence that polycystins are involved in Hydra cnidocyte discharge.

Like other cnidarians, the freshwater organism Hydra is characterized by the possession of cnidocytes (stinging cells). Most cnidocytes are located on hydra tentacles, where they are organized along with sensory cells and ganglion cells into battery complexes. The function of the battery complexes is to integrate multiple types of stimuli for the regulation of cnidocyte discharge. The molecular mechanisms controlling the discharge of cnidocytes are not yet fully understood, but it is known that discharge depends on extracellular Ca2+ and that mechanically induced cnidocyte discharge can be enhanced by the presence of prey extracts and other chemicals. Experiments in this paper show that a PKD2 (polycystin 2) transient receptor potential (TRP) channel is expressed in hydra tentacles and bases. PKD2 (TRPP) channels belong to the TRP channel superfamily and are non-selective Ca2+ channels involved in the transduction of both mechanical and chemical stimuli in other organisms. Non-specific PKD2 channel inhibitors Neo (neomycin) and Gd3+ (gadolinium) inhibit both prey capture and cnidocyte discharge in hydra. The PKD2 activator Trip (triptolide) enhances cnidocyte discharge in both starved and satiated hydra and reduces the inhibition of cnidocyte discharge caused by Neo. PKD1 and 2 proteins are known to act together to transduce mechanical and chemical stimuli; in situ hybridization experiments show that a PKD1 gene is expressed in hydra tentacles and bases, suggesting that polycystins play a direct or indirect role in cnidocyte discharge.

Do novel genes drive morphological novelty? An investigation of the nematosomes in the sea anemone Nematostella vectensis.

BACKGROUND: The evolution of novel genes is thought to be a critical component of morphological innovation but few studies have explicitly examined the contribution of novel genes to the evolution of novel tissues. Nematosomes, the free-floating cellular masses that circulate through the body cavity of the sea anemone Nematostella vectensis, are the defining apomorphy of the genus Nematostella and are a useful model for understanding the evolution of novel tissues. Although many hypotheses have been proposed, the function of nematosomes is unknown. To gain insight into their putative function and to test hypotheses about the role of lineage-specific genes in the evolution of novel structures, we have re-examined the cellular and molecular biology of nematosomes. RESULTS: Using behavioral assays, we demonstrate that nematosomes are capable of immobilizing live brine shrimp (Artemia salina) by discharging their abundant cnidocytes. Additionally, the ability of nematosomes to engulf fluorescently labeled bacteria (E. coli) reveals the presence of phagocytes in this tissue. Using RNA-Seq, we show that the gene expression profile of nematosomes is distinct from that of the tentacles and the mesenteries (their tissue of origin) and, further, that nematosomes (a Nematostella-specific tissue) are enriched in Nematostella-specific genes. CONCLUSIONS: Despite the small number of cell types they contain, nematosomes are distinct among tissues, both functionally and molecularly. We provide the first evidence that nematosomes comprise part of the innate immune system in N. vectensis, and suggest that this tissue is potentially an important place to look for genes associated with pathogen stress. Finally, we demonstrate that Nematostella-specific genes comprise a significant proportion of the differentially expressed genes in all three of the tissues we examined and may play an important role in novel cell functions.