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.

Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

Evolution of glial cells: a non-bilaterian perspective.

Nervous systems of bilaterian animals generally consist of two cell types: neurons and glial cells. Despite accumulating data about the many important functions glial cells serve in bilaterian nervous systems, the evolutionary origin of this abundant cell type remains unclear. Current hypotheses regarding glial evolution are mostly based on data from model bilaterians. Non-bilaterian animals have been largely overlooked in glial studies and have been subjected only to morphological analysis. Here, we provide a comprehensive overview of conservation of the bilateral gliogenic genetic repertoire of non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera). We overview molecular and functional features of bilaterian glial cell types and discuss their possible evolutionary history. We then examine which glial features are present in non-bilaterians. Of these, cnidarians show the highest degree of gliogenic program conservation and may therefore be crucial to answer questions about glial evolution.

Synthetic ShK-like Peptide from the Jellyfish Nemopilema nomurai Has Human Voltage-Gated Potassium-Channel-Blocking Activity.

We identified a new human voltage-gated potassium channel blocker, NnK-1, in the jellyfish Nemopilema nomurai based on its genomic information. The gene sequence encoding NnK-1 contains 5408 base pairs, with five introns and six exons. The coding sequence of the NnK-1 precursor is 894 nucleotides long and encodes 297 amino acids containing five presumptive ShK-like peptides. An electrophysiological assay demonstrated that the fifth peptide, NnK-1, which was chemically synthesized, is an effective blocker of hKv1.3, hKv1.4, and hKv1.5. Multiple-sequence alignment with cnidarian Shk-like peptides, which have Kv1.3-blocking activity, revealed that three residues (3Asp, 25Lys, and 34Thr) of NnK-1, together with six cysteine residues, were conserved. Therefore, we hypothesized that these three residues are crucial for the binding of the toxin to voltage-gated potassium channels. This notion was confirmed by an electrophysiological assay with a synthetic peptide (NnK-1 mu) where these three peptides were substituted with 3Glu, 25Arg, and 34Met. In conclusion, we successfully identified and characterized a new voltage-gated potassium channel blocker in jellyfish that interacts with three different voltage-gated potassium channels. A peptide that interacts with multiple voltage-gated potassium channels has many therapeutic applications in various physiological and pathophysiological contexts.

Sea Anemone Membrane Attack Complex/Perforin Superfamily Demonstrates an Evolutionary Transitional State between Venomous and Developmental Functions.

Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.

Evolution of myxozoan mitochondrial genomes: insights from myxobolids.

BACKGROUND: Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms. RESULTS: Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene. CONCLUSIONS: Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

Bioinformatic Prohormone Discovery in Basal Metazoans: Insights from Trichoplax.

Experimental discovery of neuropeptides and peptide hormones is a long and tedious task. Mining the genomic and transcriptomic sequence data with robust secretory peptide prediction tools can significantly facilitate subsequent experiments. We describe the application of various in silico neuropeptide discovery methods for the placozoan Trichopax adhaerens as an illustrated example and a powerful experimental paradigm for cellular and evolutionary biology. In total, 33 placozoan (neuro)peptide-like hormone precursors were found using homology-based BLAST search and repeat-based and comparative evolutionary methods. Some of the discovered precursors are homologous to insulins and RFamide precursors from Cnidaria and other animal phyla.

Catch my drift? Between-farm dispersal of biofouling waste from salmon pen net cleaning: Potential risks for fish health.

Biofouling is a serious challenge for global salmon aquaculture and farmers have to regularly clean pen nets to avoid impacts on stock health and farms' structural integrity. The removed material is released into the surrounding environment. This includes cnidarian species such as hydroids, whose nematocyst-bearing fragments can impact gill health and fish welfare. There is also increasing evidence of the association of parasites and pathogens with biofouling organisms and cleaning fragments. It is unknown whether and how far local current regimes disperse biofouling material and whether this material reaches and interacts with adjacent pens or even neighbouring farms downstream, or wild fish populations in surrounding environments. We focussed on the cnidarian hydroid Ectopleura larynx, one of the most abundant biofouling species on Norwegian aquaculture installations. Using a 3D hydrodynamic model parameterised with physical and biological properties of hydroid particles (derived via field and laboratory studies), we simulated the dispersal of net cleaning waste from two Norwegian salmon farms. Our results demonstrate that net cleaning waste is extensively dispersed throughout neighbouring pens, and even to adjacent aquaculture facilities. Salmon were exposed to concentrations of biofouling particles up to 41-fold elevated compared to background concentrations, and for up to 30.5 h. Maximum dispersal distance of hydroid particles was 5.5 km from the point of release, achieved largely within 48 h. Least-cost distance calculations show that this distance exceeds the nearest-neighbour distance of 70 % of Norway's salmon farms (654 farms). Our study provides some evidence that actions taken to manage biofouling at salmon farms may affect neighbouring farms and surrounding natural environments. The results highlight the potential risks associated with net cleaning: the dispersal of harmful cnidarian particles, associated pathogens, and non-indigenous species, thus underlining the need for novel farming or net cleaning technologies that prevent the release of potentially harmful cleaning waste.

Marine-Derived Peptides with Anti-Hypertensive Properties: Prospects for Pharmaceuticals, Supplements, and Functional Food.

Hypertension, a major health concern linked to heart disease and premature mortality, has prompted a search for alternative treatments due to side effects of existing medications. Sustainable harvesting of low-trophic marine organisms not only enhances food security but also provides a variety of bioactive molecules, including peptides. Despite comprising only a fraction of active natural compounds, peptides are ideal for drug development due to their size, stability, and resistance to degradation. Our review evaluates the anti-hypertensive properties of peptides and proteins derived from selected marine invertebrate phyla, examining the various methodologies used and their application in pharmaceuticals, supplements, and functional food. A considerable body of research exists on the anti-hypertensive effects of certain marine invertebrates, yet many species remain unexamined. The array of assessments methods, particularly for ACE inhibition, complicates the comparison of results. The dominance of in vitro and animal in vivo studies indicates a need for more clinical research in order to transition peptides into pharmaceuticals. Our findings lay the groundwork for further exploration of these promising marine invertebrates, emphasizing the need to balance scientific discovery and marine conservation for sustainable resource use.

A Comparative Analysis of the Venom System between Two Morphotypes of the Sea Anemone Actinia equina.

The current study investigates the venom-delivery system of green and red morphotypes of the sea anemone Actinia equina to disclose its potential as a source of bioactive compounds. We compared the two morphotypes using electron and optical microscopy, proteomics, and toxicity assessment on zebrafish embryos. Specialized venom-injecting cells (nematocysts) are equally distributed and found in the tentacles of both varieties. Proteomics revealed proteins of interest in both red and green Actinia, yielding the three most abundant Gene Ontology (GO) terms related to the biological processes "proteolysis", "hemolysis in another organism" and "lipid catabolic process". Neurotoxins and cytolytic toxins similar to known cnidarian toxins like PsTX-60A and AvTX-60A, for instance, were identified in both types. Extracts from green and red anemones were toxic to zebrafish embryos, with green anemone venom appearing to be more potent. The findings highlight the presence of proteinaceous toxins in A. equina and the potential for different varieties to possess distinct bioactive compounds. Notably, pore-forming toxins are suggested for molecular probes and immunotoxins, making them valuable assets for potential biotechnological and biomedical purposes.

Parallel evolution of gravity sensing.

Omnipresent gravity affects all living organisms; it was a vital factor in the past and the current bottleneck for future space exploration. However, little is known about the evolution of gravity sensing and the comparative biology of gravity reception. Here, by tracing the parallel evolution of gravity sensing, we encounter situations when assemblies of homologous modules result in the emergence of non-homologous structures with similar systemic properties. This is a perfect example to study homoplasy at all levels of biological organization. Apart from numerous practical implementations for bioengineering and astrobiology, the diversity of gravity signaling presents unique reference paradigms to understand hierarchical homology transitions to the convergent evolution of integrative systems. Second, by comparing gravisensory systems in major superclades of basal metazoans (ctenophores, sponges, placozoans, cnidarians, and bilaterians), we illuminate parallel evolution and alternative solutions implemented by basal metazoans toward spatial orientation, focusing on gravitational sensitivity and locomotory integrative systems.

Towards the Exploration and Evolution of Insulin-like Venoms in Actiniaria (Sea anemones).

Recent studies have elucidated the diversity of genes encoding venom in Sea anemones. However, most of those genes are yet to be explored in an evolutionary context. Insulin is a common peptide across metazoans and has been coopted into a predatory venom in many venomous lineages. In this study, we focus on the diversity of insulin-derived venoms in Sea anemones and on elucidating their evolutionary history. We sourced data for 34 species of Sea anemones and found sequences belonging to two venom families which have Insulin PFAM annotations. Our findings show that both families have undergone duplication events. Members of each of the independently evolving clades have consistent predicted protein structures and distinct dN/dS values. Our work also shows that sequences allied with VP302 are part of a multidomain venom contig and have experienced a secondary gain into the venom system of cuticulate Sea anemones.

Fluorescence In Situ Hybridization as a Tool for Studying the Specification and Differentiation of Cell Types in Nematostella vectensis.

The sea anemone Nematostella vectensis is a genetically tractable cnidarian species that has become a model organism for studying the evolution of developmental processes and genome regulation, resilience to fluctuations in environmental conditions, and the response to pollutants. Gene expression analyses are central to many of these studies, and in situ hybridization has been an important method for obtaining spatial information, in particular during embryonic development. Like other cnidarians, Nematostella embryos are of comparably low morphological complexity, but they possess many cell types that are dispersed throughout the tissue and originate from broad and overlapping areas. These features have made two-color fluorescence in situ hybridization an important method to determine potential co-expression of genes and to generate hypotheses for their functions in cell fate specification. We here share protocols for single and double fluorescence in situ hybridization in Nematostella and for the combination of fluorescence in situ hybridization and immunofluorescence.

SABER-FISH in Hydractinia.

In situ hybridization allows the detection of nucleic acid sequences in fixed cells and tissues. The gelatinous nature of cnidarians and Hydractinia demands extensive and exhausting protocols to detect RNA transcripts with traditional methods (e.g., colorimetric in situ hybridization). Signal amplification by exchange reaction (SABER) fluorescence in situ hybridization (FISH) enables simplifying and multiplex imaging of RNA targets in a rapid and cost-effective manner. In one enzymatic reaction, SABER-FISH uses a strand-displacing polymerase and catalytic DNA hairpin to generate FISH probes with adjustable signal amplification, allowing highly sensitive detection of nucleic acids and reducing the number of required probes. Here I describe the methodology to detect transcripts within the cells of Hydractinia by SABER-FISH in whole-mount samples.

Reduction of small-prey capture rate and collective predation in the bleached sea anemone Exaiptasiadiaphana.

Cnidarians may dominate benthic communities, as in the case of coral reefs that foster biodiversity and provide important ecosystem services. Polyps may feed by predating mesozooplantkon and large motile prey, but many species further obtain autotrophic nutrients from photosymbiosis. Anthropogenic disturbance, such as the rise of seawater temperature and turbidity, can lead to the loss of symbionts, causing bleaching. Prolonged periods of bleaching can induce mortality events over vast areas. Heterotrophy may allow bleached cnidarians to survive for long periods of time. We tested the reinforcement of heterotrophic feeding of bleached polyps of Exaiptasia diaphana fed with both small zooplantkon and large prey, in order to evaluate if heterotrophy allows this species to compensate the reduction of autotrophy. Conversely to expected, heterotrophy was higher in unbleached polyps (+54% mesozooplankton prey and +11% large prey). The increase of heterotrophic intake may not be always used as a strategy to compensate autotrophic depletion in bleached polyps. Such a resilience strategy might be more species-specific than expected.

Lepocreadiidae (Trematoda) associated with gelatinous zooplankton (Cnidaria and Ctenophora) and fishes in Australian and Japanese waters.

We examined gelatinous zooplankton from off eastern Australia for lepocreadiid trematode metacercariae. From 221 specimens of 17 species of cnidarian medusae and 218 specimens of four species of ctenophores, infections were found in seven cnidarian and two ctenophore species. Metacercariae were distinguished using cox1 mtDNA, ITS2 rDNA and morphology. We identified three species of Prodistomum Linton, 1910 [P. keyam Bray & Cribb, 1996, P. orientale (Layman, 1930), and Prodistomum Type 3], two species of Opechona Looss, 1907 [O. kahawai Bray & Cribb, 2003 and O. cf. olssoni], and Cephalolepidapedon saba Yamaguti, 1970. Two species were found in cnidarians and ctenophores, three only in cnidarians, and one only in a ctenophore. Three Australian fishes were identified as definitive hosts; four species were collected from Scomber australasicus and one each from Arripis trutta and Monodactylus argenteus. Transmission of trematodes to these fishes by ingestion of gelatinous zooplankton is plausible given their mid-water feeding habits, although such predation is rarely reported. Combined morphological and molecular analyses of adult trematodes identified two cox1 types for C. saba, three cox1 types and species of Opechona, and six cox1 types and five species of Prodistomum of which only two are identified to species. All three genera are widely distributed geographically and have unresolved taxonomic issues. Levels of distinction between the recognised species varied dramatically for morphology, the three molecular markers, and host distribution. Phylogenetic analysis of 28S rDNA data extends previous findings that species of Opechona and Prodistomum do not form monophyletic clades.

Starvation differentially affects gene expression, immunity and pathogen susceptibility across symbiotic states in a model cnidarian.

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with food limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-κB, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-κB correlated with increased immunity only in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under food scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and non-symbiotic anemone, 'defence' responses were similarly regulated in Aiptasia and the facultative coral, but not in the non-symbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways-including NF-κB-does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator.

BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

The significance of Ethel Browne's research on Hydra for the organizer concept.

This article focuses on the roots of the organizer concept, which was developed by Hans Spemann during his studies of early embryonic development in amphibians. The fundamental properties of this axis-inducing signaling center have been elucidated through pioneering molecular research by Eddy De Robertis' laboratory and other researchers. Evolutionary comparisons have disclosed the presence of this signaling center, involving the interaction of Wnt and TGF-beta signaling pathways, existed not only in vertebrates but also in basal Metazoa such as Cnidaria. - Notably, even prior to the groundbreaking experiments conducted by Hilde Mangold and Hans Spemann, Ethel Browne conducted similar transplantation experiments on Hydra polyps. They were performed under the guidance of Thomas H Morgan and in the laboratory of Edmund B Wilson. Howard Lenhoff was the first to draw connections between Ethel Browne's transplantation experiments and those of Spemann and Mangold, igniting a vivid debate on the precedence of the organizer concept and its recognition in Nobel Prize considerations. This review critically compares the experiments conducted by Spemann and Mangold with those preceding their seminal work, concluding that the organizer concept clearly builds upon earlier research aimed at understanding developmental gradients, such as in the simple model Hydra. However, these approaches were not pursued further by Morgan, who shifted his focus towards unraveling the genetic control of development in flies, an approach that ultimately revealed the molecular identity of the Spemann organizer in vertebrates.

Nationwide Aquatic Envenomations Reported to US Poison Control Centers from 2011 to 2020.

INTRODUCTION: Aquatic envenomations are common injuries along the coastal United States that pose a public health risk and can cause significant morbidity. We examined aquatic envenomation exposures that were called in to poison control centers (PCC) in the United States from 2011 to 2020. METHODS: The Association of Poison Control Center's (AAPCC) National Poison Data System was queried for all aquatic envenomations reported during the 10 y period from January 1, 2011, to December 31, 2020. Data collected included date, exposure and geographic location, patient age and sex, signs and symptoms, management setting, treatments, and clinical outcome. Duplicated records, confirmed nonexposure, and reports not originating within the United States were excluded. RESULTS: There were 8517 human aquatic envenomations reported during the study period, 62% (5243) of whom were male; 56% (4264) of patients were 30 y or younger. There were an average of 852 calls per year, with 46% of calls occurring during June to August. California, Texas, and Florida had the highest number of envenomations during the study period. Fish (61%; 5159) and Cnidaria (30%; 2519) envenomations were the most common exposures. Overall, 37% (3151) of exposures were treated in healthcare facilities, with no deaths reported. CONCLUSIONS: The highest proportion of aquatic envenomations occurred among younger males (≤30 y) during the summer months. While rarely leading to major adverse events, aquatic envenomations were commonly reported injuries to PCC and occurred in all 50 states. Poison control centers continue to be real-time sources of information and data regarding aquatic envenomation trends.