*1.7. Sea Urchins*

Sea urchins are the most abundant animals in Brazilian shores. They are also responsible for the majority of reported marine animal accidents [122]. *Echinometra lucunter*—the rock boring urchin—can be easily found in rocky shores. Human accidents are frequent and can be associated with the animal's manipulation by bathers, or by people stepping on the animals while walking on the shore. More severe cases (in terms of the number of spines punctures) can result from people being dragged onto rocky walls by wave action. Still, the most common route that the spines penetrate the skin is through the foot or hand. This event causes local inflammatory reactions, characterized by edema, erythema and pain [123,124]. Facing this problem, the authors have wondered: is this accident solely mechanical due to the spine's penetration, or does the sea urchin have a venom that contributes to the described symptoms?

To answer that question, 'toxins' from *E. lucunter* spines were extracted, immersing the excised appendices in a physiological buffer (to avoid cell lyses by osmotic shock), followed by animal inflammation test models. Authors described that the extract induces a pro-inflammatory reaction, by increasing rolling, adhered and migrated leukocytes. Moreover, the spines extract decreased the pain threshold and induced paw edema [30]. In another study, these authors were able to isolate one molecule responsible for those effects, including its partial molecular characterization [125]. However, it was clear that there was more than one single molecule eliciting such activities; therefore, the clinical observed symptoms clearly surpass the mechanical trauma aroused by spine penetration.

This mechanism is a very successful adaptation: the venom (i.e., the 'toxins') diminishes the pain threshold—making the victim more susceptible to painful stimuli—at the same time that the spines puncture the skin. As a consequence, the mechanical accident becomes more aggressive, due to this synergism (resulting in inflammation).

*E. lucunter* spines do not contain typical venom glands, in the same way venomous animals do, but it is a living structure, full of granular cells, which are most likely to produce and secrete these toxins along the entire spine, particularly at in the spine tip, a region more susceptible to mechanical stress by contact (with possible predators and aggressors) [126]. Moreover, although the spine is composed mainly of calcium and/or magnesium carbonate, the myriad of cells embedded would significantly contribute to spine regeneration. It has been demonstrated that the spine secretes cathepsins B and/or X, an enzyme associated with matrix remodeling processes, contributing to the spine growth and regeneration, but also to the toxicity.

Besides spines accidents, consumption of sea urchins may elicit undesirable/toxic effects for the consumer, as they are usually eaten raw. Therefore, these authors have investigated the coelomic fluid of *E. lucunter*, searching for toxins (pro-inflammatory molecules, in particular). A bioactive peptide, termed 'echinometrin', capable of reducing rolling cells and increasing adhered and migrated ones—concomitant to edema induction was identified. Moreover, this peptide induced mast cell degranulation, which makes us think that histamine was responsible for the observed inflammatory reaction [127]. Actually, many consumers present allergies after the consumption of raw sea urchin, and there are studies suggesting the participation of vitellogenin in such process, by increasing IgE levels [128,129]. Echinometrin is, in fact, a cryptide [130], i.e., an internal fragment of vitellogenin. Moreover, its N- and C-termini match the amino acid specificity for (the previously reported) cathepsin B/X, suggesting a local toxin generation system, in which both substrate and processing enzyme are present and ready to act.

Once the biomonitored assay reported above proved successful in the identification of one bioactive peptide, these authors decided to performed an untargeted peptidomic approach on sea urchins' peptides. The secreted peptides from *E. lucunter*, *Lytechinus variegatus* and *Arbacia lixula* were analyzed. It was possible to observe that coelomic fluids of all three species are full of peptides. On the other hand, peptides could be identified only in the spines of *L. variegatus* and *A. lixula*, whereas *E. lucunter* spines contain mainly low molecular mass compounds. Database mining suggests that some peptides may display relevant biological effects, such as antibiotic, anticancer, antiviral, phospholipase A2 inhibitor and neuroprotective properties, making sea urchin molecules a source of new therapeutic compounds [131].

#### *1.8. Mollusks*

Peptides are abundant in marine mollusks from the Gastropoda class. They are usually referred as 'conopeptides' and are responsible for prey paralysis due to their specific action on the neuromuscular ionic channels [132,133]. The genus *Conus* is a well-known source of these conopeptides. The Tox-Prot database from Uniprot/Swiss-Prot describes that 1.370 toxins are manually annotated for 117 snail species, most of them from genus *Conus* [134,135]. On the other hand, the database platform for conopeptides, ConoServer, shows that 119 *Conus* species already have at least one protein sequence/structure elucidated. Besides that, this platform shows that conopeptides can be categorized in 12 pharmacological families or in 33 cysteine frameworks. More than 2900 mature conotoxins can be found in this database [136,137].

*Conus* can be classified into three main groups, according to their feeding behavior: worm-hunting, molluscivorous and fish-hunting snails [138]. One of them—*C. regius*—a species that dwells the USA, Central America, and Brazil, including the Fernando de Noronha archipelago, has been studied by these authors [139]. As feeding behavior is often related with venom composition, the authors have investigated what would be the feeding habits of these animal, since they were not known at the time. They found that *C. regius* preferentially preys on fire-worms, thus being categorized as a vermivorous species. Authors have also evaluated the homogeneity of the venom and have determined that, regardless of gender, size and season of the year, there was no significant variation on venom composition (as determined by RP-HPLC peak area and similarity). Under these conditions, they have found the major peak, isolated and characterized it, which led to the identification of rg11a, a conotoxin presenting the cysteine pattern C-C-CC-CC-C-C and ~5 kDa [140]. Later, these authors also described α-RgIB: a 2.7 kDa peptide bearing the CC-C-C pattern, which is an antagonist of neuronal acetylcholine receptor and is capable of inducing hyperactivity in mice and breathing difficulties [141].

#### *1.9. Stingrays*

Stingrays accounted for 69% of aquatic animal accidents in Brazil from 2007 to 2013. Most cases (88.4%) were reported in the north region and correspond to accidents caused by freshwater stingrays [142].

In general, symptoms of freshwater stingray accidents include skin necrosis, edema, erythema and intense pain, mainly at the lower limbs, which are the most common accident site. Several studies have focused on the mechanism of action of stingray toxins. One explanation is the release of proinflammatory interleukins that lead to the inflammatory reaction and pain, besides the direct participation of mast cell degranulation and histamine release [143,144]. The presence of inflammatory cells in the necrotic tissues was reported, most lymphoid, CD3+ and CD4+ cells, as well as the presence of eosinophils [145].

Although less frequent, marine rays also cause human accidents, but few works report them. In this sense, some of these authors have studied *Hypanus americanum*'s mucus, searching for toxins [146]. It is noteworthy to mention that a marine stingray's whole body is covered by mucus produced by epithelial cells. Some animals possess a calcified spine ('sting') on their tail, which is covered by an epithelium that secretes mucus. This secretion is rich in molecules involved in the chemical defense and skin homeostasis maintenance, including establishing a barrier against microorganisms.

These authors observed that the mucus is labile, denaturating in function of the temperature and storage time after collection. Moreover, the classical scratching method for mucus collection results in the attainment of a mucus rich in cellular debris and, consequently, intracellular content that masks the 'actual' mucus. Authors were forced to develop a new method: the whole animal was submerged—for 40 s—in a tank containing only freshwater. After the animal was removed, the water was acidified (0.1% final concentration) and the solution was filtered. This large volume was directly pumped into the C18-RP-HPLC column via system pump 'A'. After total sample loading, standard chromatography was performed [146].

Nevertheless, the chemical nature of the mucus revealed itself to be more complex than initially imagined by those authors. Several proteins, peptides and low-molecular-mass compounds could be detected. The mucus elicits inflammatory reactions, such as edema and leukocyte recruitment in mice. The performed zymograms displayed proteolytic activity. Moreover, authors describe the antimicrobial effect of molecules fractionated from the mucus. The proteomic analyses revealed proteins that are involved in the immune response, and are very similar to the proteins related to the sting, and also similar to proteins described in fishes from Teleostei class, indicating that the epidermal secretions of

stingrays could be more related to an innate immune system than with a venom delivery system [146]. This hypothesis was recently reinforced in a work that analyzed the genomic data of a venomous fish and associated the presence of aerolysin (considered as a toxin) with the immune system [147].
