Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems.

Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) - the most common receptors of bilaterian neuropeptides - but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by 1 to 3 of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively connected by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.

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.

Cnidarians are CLOCKing in.

Studies of the starlet sea anemone provide important insights into the early evolution of the circadian clock in animals.

Antibiotics alter development and gene expression in the model cnidarian Nematostella vectensis.

Background: Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis. Methods: Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones. Results: Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments. Discussion: Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.

Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A-E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A-E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A-E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A-E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 µM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A-E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

Giant sea anemones.

Kashimoto et al. introduce the giant sea anemones, which form mutualistic relationships with anemonefish.

Designing a Secretory form of RTX-A as an Anticancer Toxin: An In Silico Approach.

BACKGROUND: Cancer is a leading cause of death and a significant public health issue worldwide. Standard treatment methods such as chemotherapy, radiotherapy, and surgery are only sometimes effective. Therefore, new therapeutic approaches are needed for cancer treatment. Sea anemone actinoporins are pore-forming toxins (PFTs) with membranolytic activities. RTX-A is a type of PFT that interacts with membrane phospholipids, resulting in pore formation. The synthesis of recombinant proteins in a secretory form has several advantages, including protein solubility and easy purification. In this study, we aimed to discover suitable signal peptides for producing RTX-A in Bacillus subtilis in a secretory form. METHODS: Signal peptides were selected from the Signal Peptide Web Server. The probability and secretion pathways of the selected signal peptides were evaluated using the SignalP server. ProtParam and Protein-sol were used to predict the physico-chemical properties and solubility. AlgPred was used to predict the allergenicity of RTX-A linked to suitable signal peptides. Non-allergenic, stable, and soluble signal peptides fused to proteins were chosen, and their secondary and tertiary structures were predicted using GOR IV and I-TASSER, respectively. The PROCHECK server performed the validation of 3D structures. RESULTS: According to bioinformatics analysis, the fusion forms of OSMY_ECOLI and MALE_ECOLI linked to RTX-A were identified as suitable signal peptides. The final proteins with signal peptides were stable, soluble, and non-allergenic for the human body. Moreover, they had appropriate secondary and tertiary structures. CONCLUSION: The signal above peptides appears ideal for rationalizing secretory and soluble RTX-A. Therefore, the signal peptides found in this study should be further investigated through experimental researches and patents.

Diversity analysis of sea anemone peptide toxins in different tissues of Heteractis crispa based on transcriptomics.

Peptide toxins found in sea anemones venom have diverse properties that make them important research subjects in the fields of pharmacology, neuroscience and biotechnology. This study used high-throughput sequencing technology to systematically analyze the venom components of the tentacles, column, and mesenterial filaments of sea anemone Heteractis crispa, revealing the diversity and complexity of sea anemone toxins in different tissues. A total of 1049 transcripts were identified and categorized into 60 families, of which 91.0% were proteins and 9.0% were peptides. Of those 1049 transcripts, 416, 291, and 307 putative proteins and peptide precursors were identified from tentacles, column, and mesenterial filaments respectively, while 428 were identified when the datasets were combined. Of these putative toxin sequences, 42 were detected in all three tissues, including 33 proteins and 9 peptides, with the majority of peptides being ShKT domain, ß-defensin, and Kunitz-type. In addition, this study applied bioinformatics approaches to predict the family classification, 3D structures, and functional annotation of these representative peptides, as well as the evolutionary relationships between peptides, laying the foundation for the next step of peptide pharmacological activity research.

Equinins as Novel Broad-Spectrum Antimicrobial Peptides Isolated from the Cnidarian Actinia equina (Linnaeus, 1758).

Sea anemones are valuable for therapeutic research as a diversified source of bioactive molecules, due to their diverse bioactive molecules linked to predation and defence mechanisms involving toxins and antimicrobial peptides. Acid extracts from Actinia equina tentacles and body were examined for antibacterial activity against Gram-positive, Gram-negative bacteria, and fungi. The peptide fractions showed interesting minimum inhibitory concentration (MIC) values (up to 0.125 µg/mL) against the tested pathogens. Further investigation and characterization of tentacle acid extracts with significant antimicrobial activity led to the purification of peptides through reverse phase chromatography on solid phase and HPLC. Broad-spectrum antimicrobial peptide activity was found in 40% acetonitrile fractions. The resulting peptides had a molecular mass of 2612.91 and 3934.827 Da and MIC ranging from 0.06 to 0.20 mg/mL. Sequencing revealed similarities to AMPs found in amphibians, fish, and Cnidaria, with anti-Gram+, Gram-, antifungal, candidacidal, anti-methicillin-resistant Staphylococcus aureus, carbapenemase-producing, vancomycin-resistant bacteria, and multi-drug resistant activity. Peptides 6.2 and 7.3, named Equinin A and B, respectively, were synthesized and evaluated in vitro towards the above-mentioned bacterial pathogens. Equinin B exerted interesting antibacterial activity (MIC and bactericidal concentrations of 1 mg/mL and 0.25 mg/mL, respectively) and gene organization supporting its potential in applied research.

Artificial light at night alters the feeding activity and two molecular indicators in the plumose sea anemone Metridium senile (L.).

Artificial light at night (ALAN) is becoming a widespread stressor in coastal ecosystems, affecting species that rely on natural day/night cycles. Yet, studies examining ALAN effects remain limited, particularly in the case of sessile species. This study assessed the effects of ALAN upon the feeding activity and two molecular indicators in the widespread plumose sea anemone Metridium senile. Anemones were exposed to either natural day/night or ALAN conditions to monitor feeding activity, and tissue samples were collected to quantify proteins and superoxide dismutase (SOD) enzyme concentrations. In day/night conditions, sea anemones showed a circadian rhythm of activity in which feeding occurs primarily at night. This rhythm was altered by ALAN, which turned it into a reduced and more uniform pattern of feeding. Consistently, proteins and SOD concentrations were significantly lower in anemones exposed to ALAN, suggesting that ALAN can be harmful to sea anemones and potentially other marine sessile species.

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.

Sea anemones, methylmercury, and bacterial infection: A closer look at multiple stressors.

Specimens of the Mediterranean sea anemone Anemonia viridis were exposed to methylmercury (MeHg) and bacterial infection to study their immune responses to a well-known toxic pollutant. Anemones were housed in laboratory conditions and divided into five experimental groups: 1. control (no microinjection); 2. filtered seawater + buffer injection; 3. filtered seawater + Escherichia coli injection; 4. MeHg + buffer injection; 5. MeHg + E. coli injection. Data showed an increase in antioxidant enzyme production compared to the constitutive condition, while methylmercury inhibited lysozyme production. The buffer inoculation had no statistically significant effects on the animals. In addition, electrophoretic and protease analyses revealed differences in the type of proteins produced, as well as a modulation of proteases depending on the treatment. The study demonstrated the immunomodulatory effect of the organic pollutant on A. viridis, validating its use as a model organism for marine coastal biomonitoring programmes and multiple stress studies.

Synthesis and Hypoglycemic Effect of Insulin from the Venom of Sea Anemone Exaiptasia diaphana.

Sea anemone venom, abundant in protein and peptide toxins, serves primarily for predatory defense and competition. This study delves into the insulin-like peptides (ILPs) present in sea anemones, particularly focusing on their role in potentially inducing hypoglycemic shock in prey. We identified five distinct ILPs in Exaiptasia diaphana, exhibiting varied sequences. Among these, ILP-Ap04 was successfully synthesized using solid phase peptide synthesis (SPPS) to evaluate its hypoglycemic activity. When tested in zebrafish, ILP-Ap04 significantly reduced blood glucose levels in a model of diabetes induced by streptozotocin (STZ) and glucose, concurrently affecting the normal locomotor behavior of zebrafish larvae. Furthermore, molecular docking studies revealed ILP-Ap04's unique interaction with the human insulin receptor, characterized by a detailed hydrogen-bonding network, which supports a unique mechanism for its hypoglycemic effects. Our findings suggest that sea anemones have evolved sophisticated strategies to activate insulin receptors in vertebrates, providing innovative insights into the design of novel drugs for the treatment of diabetes.

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.

Integrated biomarker responses in wild populations of the intertidal sea anemone Bunodosoma zamponii living under different anthropogenic pressures.

Bunodosoma zamponii is the most abundant anemone in Mar del Plata (Buenos Aires, Argentina). Given that the presence of persistent organic pollutants (organochlorine pesticides and PCBs) and the organophosphate pesticide chlorpyrifos has recently been reported in this species, two wild populations living under different anthropogenic pressures were studied and compared regarding basic aspects of their ecology and physiological response to oxidative stress. A population from an impacted site (Las Delicias, LD) and another from a reference site (Punta Cantera, PC) were monitored seasonally (spring, summer, autumn, and winter), for one year. Anemones from PC were larger and more abundant than those from LD for most sampling periods. During winter, glutathione-S-transferase and catalase activities were higher in LD. Moreover, protein content and antioxidant defenses were higher in anemones from PC during winter as well. Taking into account their ecology (size and abundance) and biomarker responses, the population from PC was comparatively healthier. Furthermore, such differences are in agreement with recent studies indicating a higher concentration of pollutants in anemones from LD (specially during the winter sampling). In this sense, considering that B. zamponii can bioaccumulate the aforementioned pollutants, its resilience to their presence, and the fact that biomarker response differed between sites, this species can be regarded as a proper sentinel species of environmental pollution. Overall, this anemone seems to be a good bioindicator to be considered in future biomonitoring and ecotoxicological studies.

Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Photosynthesis and other factors affecting the establishment and maintenance of cnidarian-dinoflagellate symbiosis.

Coral growth depends on the partnership between the animal hosts and their intracellular, photosynthetic dinoflagellate symbionts. In this study, we used the sea anemone Aiptasia, a laboratory model for coral biology, to investigate the poorly understood mechanisms that mediate symbiosis establishment and maintenance. We found that initial colonization of both adult polyps and larvae by a compatible algal strain was more effective when the algae were able to photosynthesize and that the long-term maintenance of the symbiosis also depended on photosynthesis. In the dark, algal cells were taken up into host gastrodermal cells and not rapidly expelled, but they seemed unable to reproduce and thus were gradually lost. When we used confocal microscopy to examine the interaction of larvae with two algal strains that cannot establish stable symbioses with Aiptasia, it appeared that both pre- and post-phagocytosis mechanisms were involved. With one strain, algae entered the gastric cavity but appeared to be completely excluded from the gastrodermal cells. With the other strain, small numbers of algae entered the gastrodermal cells but appeared unable to proliferate there and were slowly lost upon further incubation. We also asked if the exclusion of either incompatible strain could result simply from their cells' being too large for the host cells to accommodate. However, the size distributions of the compatible and incompatible strains overlapped extensively. Moreover, examination of macerates confirmed earlier reports that individual gastrodermal cells could expand to accommodate multiple algal cells. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

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.

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.

Graded FGF activity patterns distinct cell types within the apical sensory organ of the sea anemone Nematostella vectensis.

Bilaterian animals have evolved complex sensory organs comprised of distinct cell types that function coordinately to sense the environment. Each sensory unit has a defined architecture built from component cell types, including sensory cells, non-sensory support cells, and dedicated sensory neurons. Whether this characteristic cellular composition is present in the sensory organs of non-bilaterian animals is unknown. Here, we interrogate the cell type composition and gene regulatory networks controlling development of the larval apical sensory organ in the sea anemone Nematostella vectensis. Using single cell RNA sequencing and imaging approaches, we reveal two unique cell types in the Nematostella apical sensory organ, GABAergic sensory cells and a putative non-sensory support cell population. Further, we identify the paired-like (PRD) homeodomain gene prd146 as a specific sensory cell marker and show that Prd146+ sensory cells become post-mitotic after gastrulation. Genetic loss of function approaches show that Prd146 is essential for apical sensory organ development. Using a candidate gene knockdown approach, we place prd146 downstream of FGF signaling in the apical sensory organ gene regulatory network. Further, we demonstrate that an aboral FGF activity gradient coordinately regulates the specification of both sensory and support cells. Collectively, these experiments define the genetic basis for apical sensory organ development in a non-bilaterian animal and reveal an unanticipated degree of complexity in a prototypic sensory structure.