CLOCK evolved in cnidaria to synchronize internal rhythms with diel environmental cues.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.

A new invertebrate NPY-like polypeptide, ZoaNPY, from the Zoanthus sociatus, as a novel ligand of human NPY Y2 receptor rescues vascular insufficiency via PLC/PKC and Src- FAK-dependent signaling pathways.

Our recent multi-omics studies have revealed rich sources of novel bioactive proteins and polypeptides from marine organisms including cnidarians. In the present study, we initially conducted a transcriptomic analysis to review the composition profile of polypeptides from Zoanthus sociatus. Then, a newly discovered NPY-like polypeptide-ZoaNPY was selected for further in silico structural, binding and virtually pharmacological studies. To evaluate the pro-angiogenic effects of ZoaNPY, we employed an in vitro HUVECs model and an in vivo zebrafish model. Our results indicate that ZoaNPY, at 1-100 pmol, enhances cell survival, migration and tube formation in the endothelial cells. Besides, treatment with ZoaNPY could restore a chemically-induced vascular insufficiency in zebrafish embryos. Western blot results demonstrated the application of ZoaNPY could increase the phosphorylation of proteins related to angiogenesis signaling including PKC, PLC, FAK, Src, Akt, mTOR, MEK, and ERK1/2. Furthermore, through molecular docking and surface plasmon resonance (SPR) verification, ZoaNPY was shown to directly and physically interact with NPY Y2 receptor. In view of this, all evidence showed that the pro-angiogenic effects of ZoaNPY involve the activation of NPY Y2 receptor, thereby activating the Akt/mTOR, PLC/PKC, ERK/MEK and Src- FAK-dependent signaling pathways. Furthermore, in an excision wound model, the treatment with ZoaNPY was shown to accelerate the wound healing process in mice. Our findings provide new insights into the discovery and development of novel pro-angiogenic drugs derived from NPY-like polypeptides in the future.

Giants among Cnidaria: Large Nuclear Genomes and Rearranged Mitochondrial Genomes in Siphonophores.

Siphonophores (Cnidaria: Hydrozoa) are abundant predators found throughout the ocean and are important constituents of the global zooplankton community. They range in length from a few centimeters to tens of meters. They are gelatinous, fragile, and difficult to collect, so many aspects of the biology of these roughly 200 species remain poorly understood. To survey siphonophore genome diversity, we performed Illumina sequencing of 32 species sampled broadly across the phylogeny. Sequencing depth was sufficient to estimate nuclear genome size from k-mer spectra in six specimens, ranging from 0.7 to 2.3 Gb, with heterozygosity estimates between 0.69% and 2.32%. Incremental k-mer counting indicates k-mer peaks can be absent with nearly 20× read coverage, suggesting minimum genome sizes range from 1.4 to 5.6 Gb in the 25 samples without peaks in the k-mer spectra. This work confirms most siphonophore nuclear genomes are large relative to the genomes of other cnidarians, but also identifies several with reduced size that are tractable targets for future siphonophore nuclear genome assembly projects. We also assembled complete mitochondrial genomes for 33 specimens from these new data, indicating a conserved gene order shared among nonsiphonophore hydrozoans, Cystonectae, and some Physonectae, revealing the ancestral mitochondrial gene order of siphonophores. Our results also suggest extensive rearrangement of mitochondrial genomes within other Physonectae and in Calycophorae. Though siphonophores comprise a small fraction of cnidarian species, this survey greatly expands our understanding of cnidarian genome diversity. This study further illustrates both the importance of deep phylogenetic sampling and the utility of k-mer-based genome skimming in understanding the genomic diversity of a clade.

Physiological and transcriptomic responses of Aurelia coerulea polyps to acidified seawater conditions.

Scyphozoan jellyfish, known for their evolutionary position and ecological significance, are thought to exhibit relatively notable resilience to ocean acidification. However, knowledge regarding the molecular mechanisms underlying the scyphozoan jellyfish response to acidified seawater conditions is currently lacking. In this study, two independent experiments were conducted to determine the physiological and molecular responses of moon jellyfish (Aurelia coerulea) polyps to within- and trans-generational exposure to two reduced pH treatments (pH 7.8 and pH 7.6). The results revealed that the asexual reproduction of A. coerulea polyps significantly declined under acute exposure to pH 7.6 compared with that of polyps at ambient pH conditions. Transcriptomics revealed a notable upregulation of genes involved in immunity and cytoskeleton components. In contrast, genes associated with metabolism were downregulated in response to reduced pH treatments after 6 weeks of within-generational acidified conditions. However, reduced pH treatments had no significant influence on the asexual reproduction of A. coerulea polyps after exposure to acidified conditions over a total of five generations, suggesting that A. coerulea polyps may acclimate to low pH levels. Transcriptomics revealed distinct gene expression profiles between within- and trans-generational exposure groups to two reduced pH treatments. The offspring polyps of A. coerulea subjected to trans-generational acidified conditions exhibited both upregulated and downregulated expression of genes associated with metabolism. These physiological and transcriptomic characteristics of A. coerulea polyps in response to elevated CO2 levels suggest that polyps produced asexually under acidified conditions may be resilient to such conditions in the future.

Evolutionary Analysis of Cnidaria Small Cysteine-Rich Proteins (SCRiPs), an Enigmatic Neurotoxin Family from Stony Corals and Sea Anemones (Anthozoa: Hexacorallia).

Cnidarians (corals, sea anemones, and jellyfish) produce toxins that play central roles in key ecological processes, including predation, defense, and competition, being the oldest extant venomous animal lineage. Cnidaria small cysteine-rich proteins (SCRiPs) were the first family of neurotoxins detected in stony corals, one of the ocean's most crucial foundation species. Yet, their molecular evolution remains poorly understood. Moreover, the lack of a clear classification system has hindered the establishment of an accurate and phylogenetically informed nomenclature. In this study, we extensively surveyed 117 genomes and 103 transcriptomes of cnidarians to identify orthologous SCRiP gene sequences. We annotated a total of 168 novel putative SCRiPs from over 36 species of stony corals and 12 species of sea anemones. Phylogenetic reconstruction identified four distinct SCRiP subfamilies, according to strict discrimination criteria based on well-supported monophyly with a high percentage of nucleotide and amino acids' identity. Although there is a high prevalence of purifying selection for most SCRiP subfamilies, with few positively selected sites detected, a subset of Acroporidae sequences is influenced by diversifying positive selection, suggesting potential neofunctionalizations related to the fine-tuning of toxin potency. We propose a new nomenclature classification system relying on the phylogenetic distribution and evolution of SCRiPs across Anthozoa, which will further assist future proteomic and functional research efforts.

A new look at the architecture and dynamics of the Hydra nerve net.

The Hydra nervous system is the paradigm of a 'simple nerve net'. Nerve cells in Hydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM (transmission electron microscopy) and serial block face SEM (scanning electron microscopy) show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.

Marine Antimicrobial Peptides: An Emerging Nightmare to the Life-Threatening Pathogens.

The emergence of multidrug-resistant pathogens due to improper usage of conventional antibiotics has created a global health crisis. Alternatives to antibiotics being an urgent need, the scientific community is forced to search for new antimicrobials. This exploration has led to the discovery of antimicrobial peptides, a group of small peptides occurring in different phyla such as Porifera, Cnidaria, Annelida, Arthropoda, Mollusca, Echinodermata, and Chordata, as a component of their innate immune system. The marine environment, possessing immense diversity of organisms, is undoubtedly one of the richest sources of unique potential antimicrobial peptides. The distinctiveness of marine antimicrobial peptides lies in their broad-spectrum activity, mechanism of action, less cytotoxicity, and high stability, which form the benchmark for developing a potential therapeutic. This review aims to (1) synthesise the available information on the distinctive antimicrobial peptides discovered from marine organisms, particularly over the last decade, and (2) discuss the distinctiveness of marine antimicrobial peptides and their prospects.

The impact of food availability on tumorigenesis is evolutionarily conserved.

The inability to control cell proliferation results in the formation of tumors in many multicellular lineages. Nonetheless, little is known about the extent of conservation of the biological traits and ecological factors that promote or inhibit tumorigenesis across the metazoan tree. Particularly, changes in food availability have been linked to increased cancer incidence in humans, as an outcome of evolutionary mismatch. Here, we apply evolutionary oncology principles to test whether food availability, regardless of the multicellular lineage considered, has an impact on tumorigenesis. We used two phylogenetically unrelated model systems, the cnidarian Hydra oligactis and the fish Danio rerio, to investigate the impact of resource availability on tumor occurrence and progression. Individuals from healthy and tumor-prone lines were placed on four diets that differed in feeding frequency and quantity. For both models, frequent overfeeding favored tumor emergence, while lean diets appeared more protective. In terms of tumor progression, high food availability promoted it, whereas low resources controlled it, but without having a curative effect. We discuss our results in light of current ideas about the possible conservation of basic processes governing cancer in metazoans (including ancestral life history trade-offs at the cell level) and in the framework of evolutionary medicine.

Associative learning: Box jellyfish learns to avoid bumps.

Operant conditioning - learning to do something for a desired outcome - has never been convincingly demonstrated in Cnidaria. A study now shows that box jellyfish, Tripedalia cystophora, can learn to avoid bumping into an obstacle based on visual cues.

A remote sensing approach for exploring the dynamics of jellyfish, relative to the water current.

Drifting in large numbers, jellyfish often interfere in the operation of nearshore electrical plants, cause disturbances to marine recreational activity, encroach upon local fish populations, and impact food webs. Understanding the dynamic mechanisms behind jellyfish behavior is of importance in order to create migration models. In this work, we focus on the small-scale dynamics of jellyfish and offer a novel method to accurately track the trajectory of individual jellyfish with respect to the water current. The existing approaches for similar tasks usually involve a surface float tied to the jellyfish for location reference. This operation may induce drag on the jellyfish, thereby affecting its motion. Instead, we propose to attach an acoustic tag to the jellyfish's bell and then track its geographical location using acoustic beacons, which detect the tag's emissions, decode its ID and depth, and calculate the tag's position via time-difference-of-arrival acoustic localization. To observe the jellyfish's motion relative to the water current, we use a submerged floater that is deployed together with the released tagged jellyfish. Being Lagrangian on the horizontal plane while maintaining an on-demand depth, the floater drifts with the water current; thus, its trajectory serves as a reference for the current's velocity field. Using an acoustic modem and a hydrophone mounted to the floater, the operator from the deploying boat remotely changes the depth of the floater on-the-fly, to align it with that of the tagged jellyfish (as reported by the jellyfish's acoustic tag), thereby serving as a reference for the jellyfish's 3D motion with respect to the water current. We performed a proof-of-concept to demonstrate our approach over three jellyfish caught and tagged in Haifa Bay, and three corresponding floaters. The results present different dynamics for the three jellyfish, and show how they can move with, and even against, the water current.

Reproductive and environmental traits explain the variation in egg size among Medusozoa (Cnidaria).

Medusozoa (Cnidaria) are characterized by diverse life cycles, with different semaphoronts (medusa, medusoid, fixed gonophore, polyp) representing the sexual phase and carrying the gametes. Although egg size is often considered a proxy to understand reproductive and developmental traits of medusozoans, understanding of the processes influencing egg size variation in the group under an evolutionary context is still limited. We carried out a comprehensive review of the variation of egg size in Medusozoa to test whether this variation is related to biological/sexual or environmental traits. Egg size presents a strong phylogenetic signal (λ = 0.79, K = 0.67), explaining why closely related species with different reproductive strategies and different individual sizes have similar egg sizes. However, variation in egg size is influenced by the number of eggs, depth and temperature, with larger eggs frequently present in species with few eggs (1-15), in deep-sea species and in cold-water species. Conversely, the production of small eggs among cold-water species of Staurozoa might be associated with the development of a small benthic larvae in this group. Our study reinforces that egg sizes respond to reproductive and environmental traits, although egg size is highly conserved within medusa classes.

Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa).

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.

The Zooxanthellate Jellyfish Holobiont Cassiopea andromeda, a Source of Soluble Bioactive Compounds.

Cassiopea andromeda (Forsskål, 1775), commonly found across the Indo-Pacific Ocean, the Red Sea, and now also in the warmest areas of the Mediterranean Sea, is a scyphozoan jellyfish that hosts autotrophic dinoflagellate symbionts (family Symbiodiniaceae). Besides supplying photosynthates to their host, these microalgae are known to produce bioactive compounds as long-chain unsaturated fatty acids, polyphenols, and pigments, including carotenoids, with antioxidant properties and other beneficial biological activities. By the present study, a fractionation method was applied on the hydroalcoholic extract from two main body parts (oral arms and umbrella) of the jellyfish holobiont to obtain an improved biochemical characterization of the obtained fractions from the two body parts. The composition of each fraction (i.e., proteins, phenols, fatty acids, and pigments) as well as the associated antioxidant activity were analyzed. The oral arms proved richer in zooxanthellae and pigments than the umbrella. The applied fractionation method was effective in separating pigments and fatty acids into a lipophilic fraction from proteins and pigment-protein complexes. Therefore, the C. andromeda-dinoflagellate holobiont might be considered as a promising natural source of multiple bioactive compounds produced through mixotrophic metabolism, which are of interest for a wide range of biotechnological applications.

Apoptotic gene loss in Cnidaria is associated with transition to parasitism.

The phylum Cnidaria consists of several morphologically diverse classes including Anthozoa, Cubozoa, Hydrozoa, Polypodiozoa, Scyphozoa, Staurozoa, and Myxozoa. Myxozoa comprises two subclasses of obligate parasites-Myxosporea and Malacosporea, which demonstrate various degrees of simplification. Myxosporea were previously reported to lack the majority of core protein domains of apoptotic proteins including caspases, Bcl-2, and APAF-1 homologs. Other sequenced Cnidaria, including the parasite Polypodium hydriforme from Polypodiozoa do not share this genetic feature. Whether this loss of core apoptotic proteins is unique to Myxosporea or also present in its sister subclass Malacosporea was not previously investigated. We show that the presence of core apoptotic proteins gradually diminishes from free-living Cnidaria to Polypodium to Malacosporea to Myxosporea. This observation does not favor the hypothesis of catastrophic simplification of Myxosporea at the genetic level, but rather supports a stepwise adaptation to parasitism that likely started from early parasitic ancestors that gave rise to Myxozoa.

Peptide-driven control of somersaulting in Hydra vulgaris.

The cnidarian Hydra vulgaris has a simple nervous system with a few hundred neurons in distributed networks. Yet Hydra can perform somersaults, a complex acrobatic locomotion. To understand the neural mechanisms of somersaulting we used calcium imaging and found that rhythmical potential 1 (RP1) neurons activate before somersaulting. Decreasing RP1 activity or ablating RP1 neurons reduced somersaulting, while two-photon activation of RP1 neurons induced somersaulting. Hym-248, a peptide synthesized by RP1 cells, selectively generated somersaulting. We conclude that RP1 activity, via release of Hym-248, is necessary and sufficient for somersaulting. We propose a circuit model to explain the sequential unfolding of this locomotion, using integrate-to-threshold decision making and cross-inhibition. Our work demonstrates that peptide-based signaling is used by simple nervous systems to generate behavioral fixed action patterns. VIDEO ABSTRACT.

Protective Effects of Epigallocatechin-3-gallate (EGCG) against the Jellyfish Nemopilema nomurai Envenoming.

Jellyfish stings are the most common marine animal injuries worldwide, with approximately 150 million envenomation cases annually, and the victims may suffer from severe pain, itching, swelling, inflammation, arrhythmias, cardiac failure, or even death. Consequently, identification of effective first aid reagents for jellyfish envenoming is urgently needed. Here, we found that the polyphenol epigallocatechin-3-gallate (EGCG) markedly antagonized the hemolytic toxicity, proteolytic activity, and cardiomyocyte toxicity of the jellyfish Nemopilema nomurai venom in vitro and could prevent and treat systemic envenoming caused by N. nomurai venom in vivo. Moreover, EGCG is a natural plant active ingredient and widely used as a food additive without toxic side effects. Hence, we suppose that EGCG might be an effective antagonist against systemic envenoming induced by jellyfish venom.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Chemical Compositions and Experimental and Computational Modeling of the Anticancer Effects of Cnidocyte Venoms of Jellyfish Cassiopea andromeda and Catostylus mosaicus on Human Adenocarcinoma A549 Cells.

Nowadays, major attention is being paid to curing different types of cancers and is focused on natural resources, including oceans and marine environments. Jellyfish are marine animals with the ability to utilize their venom in order to both feed and defend. Prior studies have displayed the anticancer capabilities of various jellyfish. Hence, we examined the anticancer features of the venom of Cassiopea andromeda and Catostylus mosaicus in an in vitro situation against the human pulmonary adenocarcinoma (A549) cancer cell line. The MTT assay demonstrated that both mentioned venoms have anti-tumoral ability in a dose-dependent manner. Western blot analysis proved that both venoms can increase some pro-apoptotic factors and reduce some anti-apoptotic molecules that lead to the inducing of apoptosis in A549 cells. GC/MS analysis demonstrated some compounds with biological effects, including anti-inflammatory, antioxidant and anti-cancer activities. Molecular docking and molecular dynamic showed the best position of each biologically active component on the different death receptors, which are involved in the process of apoptosis in A549 cells. Ultimately, this study has proven that both venoms of C. andromeda and C. mosaicus have the capability to suppress A549 cells in an in vitro condition and they might be utilized in order to design and develop brand new anticancer agents in the near future.

Negative and positive interspecific interactions involving jellyfish polyps in marine sessile communities.

Sessile marine invertebrates on hard substrates are one of the two canonical examples of communities structured by competition, but some aspects of their dynamics remain poorly understood. Jellyfish polyps are an important but under-studied component of these communities. We determined how jellyfish polyps interact with their potential competitors in sessile marine hard-substrate communities, using a combination of experiments and modelling. We carried out an experimental study of the interaction between polyps of the moon jellyfish Aurelia aurita and potential competitors on settlement panels, in which we determined the effects of reduction in relative abundance of either A. aurita or potential competitors at two depths. We predicted that removal of potential competitors would result in a relative increase in A. aurita that would not depend on depth, and that removal of A. aurita would result in a relative increase in potential competitors that would be stronger at shallower depths, where oxygen is less likely to be limiting. Removal of potential competitors resulted in a relative increase in A. aurita at both depths, as predicted. Unexpectedly, removal of A. aurita resulted in a relative decrease in potential competitors at both depths. We investigated a range of models of competition for space, of which the most successful involved enhanced overgrowth of A. aurita by potential competitors, but none of these models was completely able to reproduce the observed pattern. Our results suggest that interspecific interactions in this canonical example of a competitive system are more complex than is generally believed.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica.

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.