*1.10. Cnidarians*

The phylum Cnidaria comprises more than 10,000 species and is considered the most ancient venomous animal lineage, having emerged approximately 650 million years ago [148,149]. To the contrary of other venomous animals, cnidarians have the unique characteristic of lacking a centralized venom system [150]. Instead of a venom gland, these animals present little organelles distributed throughout their bodies, called cnidaes. Such structures are produced by the Golgi apparatus of specialized cells: the cnidoblasts [151]. It is divided into three main lineages: 1. Anthozoa, formed by Anthozoa class; 2. Medusozoa, comprised of Scyphozoa, Staurozoa, Cubozoa and Hydrozoa classes; 3. Endocnidozoa, comprising Myxozoa and Polypodiozoa classes. Cnidaria is a diverse phylum, rich in bioactive molecules, known to be used mainly for predation, defense and intraspecific competition [152].

Cnidaria early studies began in 1903 on *Anemonia sulcata* and *Actinia equina* tentacles extracts. Since then, several studies on sea anemones have been developed, leading to more than a century of research on these animals' venoms [150,153,154]. Sea anemones are exquisite sources of toxins and represent the greatest diversity in Anthozoa, having around 1200 species distributed in 46 families [150].

These cnidarians can cause envenomation through their nematocysts, specialized structures that inoculate venom. One particular case report of a human accident caused by anemones belonging to the *Stichodactyla* genus describes local skin irritations with blistering, edema and hemorrhage, mild symptoms when compared to the actual target of the toxin, prey, which is instantly killed by neuro- and cardiotoxins [155]. The anemone toxins molecular scaffolds are diverse: at least 17 different structural motifs are known [150].

The peptide neurotoxins found in sea anemones may act over different ion channels. ShK toxins, for example, bind to Kv type 1; some types of β-defensins can modify the action of Kv type 3 and Nav type 1, 2 and 4; while the inhibitor cystine-knot (ICK) can act over Kv type 5 and acid-sensing ion channels [150]. In this context, a study published in 2004, by some of these authors, investigated the differential selectivity between three sea anemones toxins against a wide range of Nav channels subtypes (Nav 1.1–1.6). The authors observed that for Nav1.3, the three toxins (ATX-II, AFT-II and Bc-III) were active only when at high concentrations. Additionally, it was observed that although ATX-II (from *A. sulcata*) and AFT-II (from *A. fuscoviridis*) exhibit similar sequences, a single amino difference was enough to alter the ion channels specificity. Lysine36 (ATX-II) seems to be fundamental for its action over Nav1.1 and Nav1.2 channels; meanwhile, AFT-II mainly exerts effects on Nav 1.4 and Nav 1.5. Moreover, the slight changes in amino acids between similar Nav channels can have a crucial role in toxins binding. For example, AFT-II had a more potent effect over Nav1.4 than Nav 1.5. These two channels are only marginally different and the presence of a Leucine at position 1611 in Nav1.4, instead of an Isoleucine at in Nav1.5 right after a neighboring Asparagine, may indicate the importance of these residues for the toxin binding [156].

In another evaluation of sea anemones venoms, Zaharenko et al. reported, for the first time, the proteomics analysis of the neurotoxic fraction of the sea anemone *Bunodosoma cangicum*. Authors processed by RP-HPLC such a fraction and identified at least 81 different molecules, distributed along 41 chromatographic peaks. Mass spectrometric analysis by MALDI-TOF and ESI-Q-TOF shows that that fraction is composed of low-molecular-mass (280–450 Da) as well as heavier molecules (4–5 kDa). Major fractions were purified and sequenced by Edman degradation, revealing nine novel peptides. Three peptides clearly presented the typical cysteine scaffold found in type 1 sodium channel toxins, and six of them presented new cysteine scaffolds belonging to two new classes of toxins. Additionally, when tested on extracellular crab leg nerve, the new peptides called Bcg31.16 and Bcg30.24 showed that, at very low concentrations (40–50 nM), those neurotoxins were able to diminish the amplitude of CAPs (compound action potentials) and increase its duration, showing a high potency and suggesting that these toxins target sodium channels [157].

Compared to other cnidarians, the Anthozoa (anemones included) is a well-studied group, in terms of toxins investigation. ToxProt lists 256 toxins belonging to 48 species of sea anemones (manually curated; accessed October, 2021). On the other hand, only five toxins from Cubozoa; four from Hydrozoa and one from Scyphozoa classes are deposited [134]. Of particular interest, three Cubozoa toxins (caTX-A, cqTX-A, crTX-A, cfTX-1 and cfTX-2) belong respectively to four species of box jellyfishes: *Carybdea alata*, *Chiropsoides quadrigatus*, *Carybdea rastonii*; and *Chironex fleckeri* (the Australian box jellyfish, one of the most dangerous species of cnidarians) [134,158]. Regardless of the small number of curated toxins, 327 proteins from Cubozoa—computationally analyzed and available at TrEMBL—still remain to be reviewed. The literature refers to Cubozoa toxins being enzymes (phospholipases A2, metallopeptidases and serine peptidases), CRISPs, lectins, pore-forming toxins and protease inhibitors [159]. For Hydrozoa, the four proteins manually curated and described as Hydralysin toxins belong to only two different species: *Hydra viridissima* and *H. vulgaris* [134,135].

The challenge of better knowing the toxins found in Cubozoa and Hydrozoa is not limited to the proteins and peptides; little is known about the low-molecular-mass molecules from these organisms [160]. In order to increase knowledge on the biotechnological potential of Cubozoa and Hydrozoa, two studies were recently performed. The first one, conducted by Bueno et al. [161], investigated the effects of the methanolic extracts of hydromedusa *Olindias sambaquiensis* and jellyfish *Chiropsalmus quadrumanus* over the autonomic neurotransmission. In this study, researchers employed a classical model to sympathetic co-transmission: a myographic evaluation of rat vas deferens bisected in two portions (prostatic and epididymal) for purinergic or adrenergic responses. Throughout the study, both methanolic extracts were demonstrated to be of low complexity and rich in low molecular mass molecules.

Authors report that a low concentration (0.1 μg/mL) of *C. quadrumanus* extract blocked the predominantly noradrenergic contraction of the epididymal end. On the other hand, only high concentrations (1 and 10 μg/mL) of *O. sambaquiensis* extract were capable of leading to the blockade of muscle contraction. Nevertheless, both extracts did not present significant differences concerning the phasic contractions in the prostatic portion (purinergic response), when compared to the control group. Moreover, the histological analysis showed that none of the extracts promote major tissue damage in the prostatic and epididymal vas deferens ends, showing the same unaltered morphology as the control group, which indicates their effects only on the neurotransmission, not causing toxic tissue damages [161].

Another study, published by Arruda et al. [160], focused on *C. quadrumanus* tentacles methanolic extract and its biological activity over neurite growth. In this work, the extract was tested on a human SH-SY5Y neuroblastoma cell line, a neuronal cell culture model commonly used for neurodegenerative disease investigations. Authors report alterations on neurite-related structures of neurons, without affecting cell proliferation or inducing necrosis or apoptosis [160]. The specific neurite length outgrowth observed in all cells exposed to the toxins was associated with a translin-like protein (hyccin cryptein) cryptide, as well as to small molecules acting synergically to promote the neurite/branches formation, elongation and facilitating neurotransmission. Neurite formation can happen either via microtubule and motor proteins [162] or PI4P regulation—acting on plasma membrane identity and myelin development [162]. Moreover, toxins present in the methanolic extract showed no effect on the straightness of neurite's growth or cell body area, but increased branching junctions connected to cells. More than 14 low molecular mass molecules related to neuritogenesis were found through LC-MS fingerprinting and at least 4 peptides related to neuronal function [160].
