Cnidarian Venoms Research Articles

Electrophysiological evaluation of the effect of peptide toxins on voltage-gated ion channels: a scoping review on theoretical and methodological aspects with focus on the Central and South American experience.

The effect of peptide toxins on voltage-gated ion channels can be reliably assessed using electrophysiological assays, such as the patch-clamp technique. However, much of the toxinological research done in Central and South America aims at purifying and characterizing biochemical properties of the toxins of vegetal or animal origin, lacking electrophysiological approaches. This may happen due to technical and infrastructure limitations or because researchers are unfamiliar with the techniques and cellular models that can be used to gain information about the effect of a molecule on ion channels. Given the potential interest of many research groups in the highly biodiverse region of Central and South America, we reviewed the most relevant conceptual and methodological developments required to implement the evaluation of the effect of peptide toxins on mammalian voltage-gated ion channels using patch-clamp. For that, we searched MEDLINE/PubMed and SciELO databases with different combinations of these descriptors: "electrophysiology", "patch-clamp techniques", "Ca2+ channels", "K+ channels", "cnidarian venoms", "cone snail venoms", "scorpion venoms", "spider venoms", "snake venoms", "cardiac myocytes", "dorsal root ganglia", and summarized the literature as a scoping review. First, we present the basics and recent advances in mammalian voltage-gated ion channel's structure and function and update the most important animal sources of channel-modulating toxins (e.g. cnidarian and cone snails, scorpions, spiders, and snakes), highlighting the properties of toxins electrophysiologically characterized in Central and South America. Finally, we describe the local experience in implementing the patch-clamp technique using two models of excitable cells, as well as the participation in characterizing new modulators of ion channels derived from the venom of a local spider, a toxins' source less studied with electrophysiological techniques. Fostering the implementation of electrophysiological methods in more laboratories in the region will strengthen our capabilities in many fields, such as toxinology, toxicology, pharmacology, natural products, biophysics, biomedicine, and bioengineering.

A Sea Anemone Lebrunia neglecta Venom Fraction Decreases Boar Sperm Cells Capacitation: Possible Involvement of HVA Calcium Channels.

Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells’ current kinetics and current–voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.

Implications of bleaching on cnidarian venom ecology.

Cnidarian bleaching research often focuses on the effects on a cnidarian's physiological health and fitness, whilst little focus has been towards the impacts of these events on their venom ecology. Given the importance of a cnidarian's venom to their survival and the increasing threat of bleaching events, it is important to understand the effects that this threat may have on this important aspect of their ecology as it may have unforeseen impacts on their ability to catch prey and defend themselves. This review aims to explore evidence that suggests that bleaching may impact on each of the key aspects of a cnidarians' venom ecology: cnidae, venom composition, and venom toxicity. Additionally, the resulting energy deficit, compensatory heterotrophic feeding, and increased defensive measures have been highlighted as possible ecological factors driving these changes. Suggestions are also made to guide the success of research in this field into the future, specifically in regards to selecting a study organism, the importance of accurate symbiont and cnidae identification, use of appropriate bleaching methods, determination of bleaching, and animal handling. Ultimately, this review highlights a significant and important gap in our knowledge into how cnidarians are, and will, continue to be impacted by bleaching stress.

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection.

BackgroundCnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known.MethodsWe searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals.ResultsWe identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish’s gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a “venom” can have and represent targets for designing therapeutics against myxozoan infections.

Cnidarians as a potential source of antiparasitic drugs

New antiparasitic drugs are urgently required for treating parasitic infections. The marine environment has proven to be a valuable source of compounds with therapeutic properties against many diseases, including parasitic diseases. Cnidarian venoms are known for their toxicological properties and are candidates for developing medications. In this review, the antiparasitic properties of cnidarian toxins, discovered over the last two decades, were examined. A total of 61 cnidarian compounds from 18 different genera of cnidaria were studied for their antiparasitic activities. The assessed genera belonged mainly to three geographical areas: South America, North America, and Southeast Asia. The in vitro activities of crude extracts and compounds against a range of parasites including Plasmodium falciparum, Trypanosoma brucei gambiense, T. cruzi, T. congolense, Leishmania donovani, L. chagasi, L. braziliensis, and Giardia duodenalis are reviewed. The challenges involved in developing these compounds into effective drugs are discussed.

The influence of ecological factors on cnidarian venoms.

Venom research is often focussed on medical relevance, novel compounds and venom evolution, whilst studying the relationship between a venom and its environment – venom ecology - has been conducted to a lesser extent. Given the projected environmental changes envisioned to occur with global warming, it is pertinent now more than ever, to highlight this topic. Here we review literature examining the influence of ecological factors such as environmental temperature, salinity, ontogeny, geographic location and diet on cnidarian venoms. This review provides an exclusive focus on the cnidarian phylum and encompasses all available published, peer-reviewed literature to our knowledge regarding the ecological factors influencing venom. We find a startling lack of research into the effects of both environmental and biological factors on venoms, with very few to no studies available per category. Importantly, research does exist that suggest these ecological processes may influence other marine or terrestrial venoms, thus we recommend future research is needed to explore this concept in cnidarians.

Inhibitory effect of plant extracts on the cytotoxicity of eurytele nematocysts from <em>Pelagia noctiluca</em>

Some species of Cnidarians, well known venomous organisms, are able to induce serious health problems as well as economic and social trouble. A lot of data show that cnidarian venoms can be cytotoxic to cultured cells. Therefore, a priority of the research is to find tools which could counteract the damaging activity of venoms. In this connection, the modulatory effects exerted by extracts of plants Ananas comosus (L.) and Carica papaya (L.) on cytotoxicity of heterotrichous microbasic eurytele nematocysts isolated from the jellyfish Pelagia noctiluca was evaluated. The nematocyst extract induced strong cytotoxicity at highest tested doses with reduction of cell survival below 40% and IC50 value of 40×103 nematocysts/mL. Both plant extracts significantly improved the survival of cells, so that the IC50 resulted 74×103 and 100×103 nematocysts/mL in the presence of A. comosus and C. papaya extracts, respectively, both used at 10 and 100 μg/mL. Taking into account that heterotrichous microbasic eurytele nematocysts are present mainly in tentacles, which Pelagia noctiluca utilizes both for predation and defence, this result is particularly interesting from both ecological and sanitary point of view and demonstrates the potential of these plants extracts against cnidarian venoms.

Ultrastructure and Molecular Toxicological Effects of the Coronate Scyphomedusa Linuche unguiculata Venom on Giardia duodenalis

Cnidarian venoms are rising as a new source of potential pharmacologically active molecules. They have been investigated for their neurotoxins acting on several nervous system targets but little is known about their antimicrobial and antiparasitic properties. Giardiasis, a common disease in developing countries, is produced by the protozoan Giardia duodenalis which induces severe gastroenteritis when infecting humans. The aim of this study is to describe the structural and molecular effects of Linuche unguiculata jellyfish extract on G. duodenalis. In this study, the effects on the ultrastructure were observed by electron transmission microscopy 24 h after the exposure to the extract and to the commercial antiparasitic drugs metronidazole, nitazoxanide and albendazole. To assess the effect at the molecular level, the gene expression analysis of β-tubulin, β-giardin, cysteine protease and glutamate dehydrogenase of G. duodenalis exposed to Linuche’s extract was performed by the RT-PCR technique. Our results show that the exposure to venom caused severe changes on Giardia’s cellular organelles such as submembrane vesicles, nuclei and adhesive disk. Our experiments also showed that the extract significantly alters the expression of genes that encode for the structural protein β-tubulin and the enzyme glutamate dehydrogenase, which are related to cell viability.

A Review of Toxins from Cnidaria.

Cnidarians have been known since ancient times for the painful stings they induce to humans. The effects of the stings range from skin irritation to cardiotoxicity and can result in death of human beings. The noxious effects of cnidarian venoms have stimulated the definition of their composition and their activity. Despite this interest, only a limited number of compounds extracted from cnidarian venoms have been identified and defined in detail. Venoms extracted from Anthozoa are likely the most studied, while venoms from Cubozoa attract research interests due to their lethal effects on humans. The investigation of cnidarian venoms has benefited in very recent times by the application of omics approaches. In this review, we propose an updated synopsis of the toxins identified in the venoms of the main classes of Cnidaria (Hydrozoa, Scyphozoa, Cubozoa, Staurozoa and Anthozoa). We have attempted to consider most of the available information, including a summary of the most recent results from omics and biotechnological studies, with the aim to define the state of the art in the field and provide a background for future research.

Transcriptomic Analysis of Four Cerianthid (Cnidaria, Ceriantharia) Venoms.

Tube anemones, or cerianthids, are a phylogenetically informative group of cnidarians with complex life histories, including a pelagic larval stage and tube-dwelling adult stage, both known to utilize venom in stinging-cell rich tentacles. Cnidarians are an entirely venomous group that utilize their proteinaceous-dominated toxins to capture prey and defend against predators, in addition to several other ecological functions, including intraspecific interactions. At present there are no studies describing the venom for any species within cerianthids. Given their unique development, ecology, and distinct phylogenetic-placement within Cnidaria, our objective is to evaluate the venom-like gene diversity of four species of cerianthids from newly collected transcriptomic data. We identified 525 venom-like genes between all four species. The venom-gene profile for each species was dominated by enzymatic protein and peptide families, which is consistent with previous findings in other cnidarian venoms. However, we found few toxins that are typical of sea anemones and corals, and furthermore, three of the four species express toxin-like genes closely related to potent pore-forming toxins in box jellyfish. Our study is the first to provide a survey of the putative venom composition of cerianthids and contributes to our general understanding of the diversity of cnidarian toxins.

An in vitro comparison of venom recovery methods and results on the box jellyfish, Chironex fleckeri

The emergence of novel venom extraction techniques over the last half-century has greatly facilitated advances in the field of cnidarian research. A new recovery protocol utilizing ethanol as the primary stimulant in nematocyst discharge was recently published, however in vitro examination of the venom on organic models was not performed. This present study reports an original comparison of the chemically-induced discharge technique in vitro with a commonly used saltwater extraction method. Size-exclusion chromatography revealed distinct differences in venom profiles between the two methods: the saltwater recovery method FPLC profile and SDS-PAGE gel were similar to previously published results, whereas the ethanol-induced method was not. SDS-PAGE gel revealed distinct 40–55 kDa bands of previously identified cardiotoxic proteins recovered from the saltwater method, whereas the ethanol-induced method yielded degraded venom protein bands. A concentration-response curve generated through xCELLigence Real-Time Cell Analysis (RTCA) revealed a dramatic decrease in human cardiomyocyte activity when venom recovered via saltwater discharge was applied to these cells. With the exception of one sample, all ethanol-induced recovered venom failed to prompt a concentration-dependent decrease in cell survival when applied to human cardiomyocytes, resulting in a significant difference in IC50 concentrations between the compared venom samples. The data presented here facilitates an improved understanding of the parameters and analyses that are essential when developing and utilizing novel techniques for future cnidarian venom extraction research and supports the conclusion that recovery of venom from the tentacles of the box jellyfish Chironex fleckeri by ethanol is not an effective, efficient, or comprehensive extraction method compared to the published method of saltwater degradation of tentacles and bead mill extraction.

Tentacle Transcriptomes of the Speckled Anemone (Actiniaria: Actiniidae: Oulactis sp.): Venom-Related Components and Their Domain Structure

Cnidarians are one of the oldest known animal lineages (ca. 700 million years), with a unique envenomation apparatus to deliver a potent mixture of peptides and proteins. Some peptide toxins from cnidarian venom have proven therapeutic potential. Here, we use a transcriptomic/proteomic strategy to identify sequences with similarity to known venom protein families in the tentacles of the endemic Australian 'speckled anemone' (Oulactis sp.). Illumina RNASeq data were assembled de novo. Annotated sequences in the library were verified by cross-referencing individuals' transcriptomes or protein expression evidence from LC-MS/MS data. Sequences include pore-forming toxins, phospholipases, peptidases, neurotoxins (sodium and potassium channel modulators), cysteine-rich secretory proteins and defensins (antimicrobial peptides). Fewer than 4% of the sequences in the library occurred across the three individuals examined, demonstrating high sequence variability of an individual's arsenal. We searched for actinoporins in Oulactis sp. to assess sequence similarity to the only described toxins (OR-A and -G) for this genus and examined the domain architecture of venom-related peptides and proteins. The novel putative actinoporin of Oulactis sp. has a greater similarity to other species in the Actiniidae family than to O. orientalis. Venom-related sequences have an architecture that occurs in single, repeat or multi-domain combinations of venom-related (e.g. ShK-like) and non-venom (e.g. whey acid protein) domains. This study has produced the first transcriptomes for an endemic Australian sea anemone species and the genus Oulactis, while identifying nearly 400 novel venom-related peptides and proteins for future structural and functional analyses and venom evolution studies.

Natural Compounds and Drug Discovery: Can Cnidarian Venom Play a Role?

Natural compounds extracted from organisms and microorganisms are an important resource for the development of drugs and bioactive molecules. Many such compounds have made valuable contributions in diverse fields such as human health, pharmaceutics and industrial applications. Presently, however, research on investigating natural compounds from marine organisms is scarce. This is somewhat surprising considering that the marine environment makes a major contribution to Earth's ecosystems and consequently possesses a vast storehouse of diverse marine species. Interestingly, of the marine bioactive natural compounds identified to date, many are venoms, coming from Cnidarians (jellyfish, sea anemones, corals). Cnidarians are therefore particularly interesting marine species, producing important biological compounds that warrant further investigation for their development as possible therapeutic agents. From an experimental aspect, this review aims to emphasize and update the current scientific knowledge reported on selected biological activity (antiinflammatory, antimicrobial, antitumoral, anticoagulant, along with several less studied effects) of Cnidarian venoms/extracts, highlighting potential aspects for ongoing research towards their utilization in human therapeutic approaches.

“Beyond Primary Sequence”—Proteomic Data Reveal Complex Toxins in Cnidarian Venoms

Venomous animals can deploy toxins for both predation and defense. These dual functions of toxins might be expected to promote the evolution of new venoms and alteration of their composition. Cnidarians are the most ancient venomous animals but our present understanding of their venom diversity is compromised by poor taxon sampling. New proteomic data were therefore generated to characterize toxins in venoms of a staurozoan, a hydrozoan, and an anthozoan. We then used a novel clustering approach to compare venom diversity in cnidarians to other venomous animals. Comparison of the presence or absence of 32 toxin protein families indicated venom composition did not vary widely among the 11 cnidarian species studied. Unsupervised clustering of toxin peptide sequences suggested that toxin composition of cnidarian venoms is just as complex as that in many venomous bilaterians, including marine snakes. The adaptive significance of maintaining a complex and relatively invariant venom remains unclear. Future study of cnidarian venom diversity, venom variation with nematocyst types and in different body regions are required to better understand venom evolution.

The Birth and Death of Toxins with Distinct Functions: A Case Study in the Sea Anemone Nematostella.

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

Comparative proteomics reveals recruitment patterns of some protein families in the venoms of Cnidaria

Cnidarians are probably the oldest group of animals to be venomous, yet our current picture of cnidarian venom evolution is highly imbalanced due to limited taxon sampling. High-throughput tandem mass spectrometry was used to determine venom composition of the scyphozoan Chrysaora lactea and two cubozoans Tamoya haplonema and Chiropsalmus quadrumanus. Protein recruitment patterns were then compared against 5 other cnidarian venom proteomes taken from the literature. A total of 28 putative toxin protein families were identified, many for the first time in Cnidaria. Character mapping analysis revealed that 17 toxin protein families with predominantly cytolytic biological activities were likely recruited into the cnidarian venom proteome before the lineage split between Anthozoa and Medusozoa. Thereafter, venoms of Medusozoa and Anthozoa differed during subsequent divergence of cnidarian classes. Recruitment and loss of toxin protein families did not correlate with accepted phylogenetic patterns of Cnidaria. Selective pressures that drive toxin diversification independent of taxonomic positioning have yet to be identified in Cnidaria and now warrant experimental consideration.

Analgesic and CNS Depressant Activities of Sea Anemone Heteractis aurora Nematocyst Toxin.

Marine organisms are the excellent sources for biologically active compounds. Cnidarian venoms are potentially valuable materials used for biomedical research and drug development. The present work was carried out to analyse haemolytic, analgesic and CNS depressant activity of sea anemone Heteractis aurora. In haemolytic assay, among the five different RBC blood cells, the chicken blood exhibited maximum hemolytic activity of 64 Hemolytic Unit (HU). The maximum Analgesic Ratio (AR) of 5 recorded at 15 and 30 min interval and minimum was recorded after 45, 60 and 120 min time intervals. In jumping response activity, the maximum of 5 AR recorded at 15, 30 & 45 min and minimum was recorded at 90 & 120 min time intervals. The maximum decrease of depressant activity of 45.07% was determined in CNS depressant activity. Anti-inflammatory activity showed significant inhibition by crude extract of Heteractis aurora.

Cnidarian Neurotoxic Peptides Affecting Central Nervous System Targets.

Natural products from animal venoms have been used widely in the discovery of novel molecules with particular biological activities that enable their use as potential drug candidates. The phylum Cnidaria (jellyfish, sea anemones, corals zoanthids, hydrozoans, etc.) is the most ancient venomous phylum on earth. Its venoms are composed of a complex mixture of peptidic compounds with neurotoxic and cytolitic properties that have shown activity on mammalian systems despite the fact that they are naturally targeted against fish and invertebrate preys, mainly crustaceans. For this reason, cnidarian venoms are an interesting and vast source of molecules with a remarkable activity on central nervous system, targeting mainly voltage-gated ion channels, ASIC channels, and TRPV1 receptors. In this brief review, we list the amino acid sequences of most cnidarian neurotoxic peptides reported to date. Additionally, we propose the inclusion of a new type of voltage-gated sea anemone sodium channel toxins based on the most recent reports.

Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: Hydrozoa)

Jellyfish venoms are of medical and biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant in Olindias spp. High throughput mass spectrometry profiling of cnidarian venoms has become increasingly popular since the first description of the proteomic profile of putative toxins isolated from nematocysts of the hydrozoan jellyfish Olindias sambaquiensis describing the presence of orthologous enzymes as presented in venoms of advanced species as snakes. Rigorous bioinformatics analyses can aid functional annotation, but biochemical assays are prerequisite to unambiguously assign toxic function to a peptide or protein. Here we present results that experimentally confirm previously predicted proteomic analysis that crude venom extracts from tentacles of O. sambaquiensis are composed of polypeptides with metalloproteinase, serine proteinase and phospholipases A2 activities. Surprisingly, levels of serine proteinase and phospholipase A2 activities were comparable to those observed in venoms of Bothrops snakes which were used as positive controls in this study. Hence, these data offer new opportunities to explore serine proteinase and phospholipase A2 activities in the clinical sequelae following O. sambaquiensis envenomation, with future possible biopharmaceutical applications.

Tentacle Transcriptome and Venom Proteome of the Pacific Sea Nettle, Chrysaora fuscescens (Cnidaria: Scyphozoa).

Jellyfish venoms are rich sources of toxins designed to capture prey or deter predators, but they can also elicit harmful effects in humans. In this study, an integrated transcriptomic and proteomic approach was used to identify putative toxins and their potential role in the venom of the scyphozoan jellyfish Chrysaora fuscescens. A de novo tentacle transcriptome, containing more than 23,000 contigs, was constructed and used in proteomic analysis of C. fuscescens venom to identify potential toxins. From a total of 163 proteins identified in the venom proteome, 27 were classified as putative toxins and grouped into six protein families: proteinases, venom allergens, C-type lectins, pore-forming toxins, glycoside hydrolases and enzyme inhibitors. Other putative toxins identified in the transcriptome, but not the proteome, included additional proteinases as well as lipases and deoxyribonucleases. Sequence analysis also revealed the presence of ShKT domains in two putative venom proteins from the proteome and an additional 15 from the transcriptome, suggesting potential ion channel blockade or modulatory activities. Comparison of these potential toxins to those from other cnidarians provided insight into their possible roles in C. fuscescens venom and an overview of the diversity of potential toxin families in cnidarian venoms.