Marine toxins are likely as diversified as the oceans’ immense biodiversity and result from co-evolutionary processes between the organisms that secrete them and their target recipients, whether prey, predators or parasites. Along with other types of natural products, these substances are considered to hold high-value bioproducts due to their potential specificity against molecular receptors and metabolic pathways. Many toxins from animals are peptides or high molecular weight proteins that interact with specific enzymes and ion channels (e.g., sodium and/or potassium ion channels), thus affecting, for example, neuromuscular, cardiovascular and immune systems (Zhang, 2015; Ponte and Modica, 2017). For these reasons, toxinology has been rising as a promising field with direct implications for drug discovery, novel therapeutic alternatives and even ecologically friendly antifoulants and pesticides. Although still lagging behind research on their terrestrial counterparts, such as snakes, bees and scorpions, marine toxinology is on the rise as researchers and bio-based industrialists become increasingly aware for the uncanny value of the oceans as an almost limitless source of novel bioproducts (see for instance Molinski et al., 2009; Leal et al., 2020).

Venoms are complex mixtures of toxins, enzymes, peptides and salts, which renders molecule isolation, identification and determination of substance-specific bioreactivity challenging. Their efficacy results from the interaction of these compounds, not just from toxins as noxious agents but also from permeabilisers like proteolytic enzymes. Still, several venomous or poisonous species of snakes, lizards, and leeches have already been used to produce toxin-based drugs for clinical applications directed against disease-induced pain (e.g., from osteoarthritis and multiple sclerosis), as anticoagulants for specific coronary interventions and also for hypertension or specific types of diabetes (see Bordon et al., 2020 for a review). Additionally, studies on wasp venoms are revealing promising alternatives for traditional antibiotics and even as antiviral and anti-seizure therapeutics (Vila-Farrés et al., 2012; Sample et al., 2013; Castro e Silva et al., 2020). In turn, pushed by the recent premises of the “Blue Growth” revolution, the last decade witnessed a growing interest in marine bioproducts, toxins included. Indeed, marine ecosystems are nowadays acknowledged to be a highly valuable source of pharmaceutically active toxins (see Greener, 2020). Notwithstanding, marine invertebrates are considered the most promising group of animals possessing toxins in face of their richness. Marine animals such cnidarians (jellyfishes, sea anemones, hydrozoans), echinoderms (starfishes, sea urchins), annelids (polychaetes), nemertines, bryozoans (moss animals), sponges, tunicates and molluscs (gastropods, cephalopods) stand out as toxin-secreting eumetazoans (see Kem, 2005; Zhang, 2015; Rodrigo and Costa, 2019). As examples, recent investigations highlighted bryostatins, which are heterocyclic molecules produced by bryozoans, and other cyclic peptides produced by endosymbiotic micro-organisms from some tunicates, as high-potential compounds for cancer therapeutics (Kem, 2005; Watters, 2018). As yet another example, clinical trials on toxic peptides from sun anemone have been revealed favourable results for the treatment of autoimmune diseases, due to their effects as strong potassium channel blockers (Prentis et al., 2018). However, the most prominent example among biotechnological applications of marine bioproducts is the approved painkiller ziconotide (commercialised as Prialt), developed from a ω-conotoxin found in the venom of the cone snail Conus magus (Williams et al., 2008). This case has recently been followed by trabectedin (Yondelis), an anti-cancer quinoline first isolated from a tropical tunicate (Cuevas and Francesch, 2009), which, albeit not primarily a toxin, yields leverage to drug development from marine bioproducts.

As more species of marine invertebrates join the ranks of toxin-secreting marine animals, attention is turning to one of the oldest-known venomous group of molluscs, the cephalopods. Perhaps surprisingly, only a few toxins have yet been described for these predators but the broad range of bioactive compounds in their venomous saliva, which includes neurotoxic proteins and peptides, amines and permeabilising enzymes, yields high promises for marine bioprospecting (see Cooke et al., 2017). Recent advances in marine “venomics” (see von Reumont et al., 2014) can shed new lights on these obscure toxins. Cephalotoxins, in particular, appear to be a unique class of toxins whose exact nature remains elusive even though their discovery dates from the mid-XX century. In this review we will summarise the scant knowledge on these toxins that are produced by the salivary glands of cephalopods, endeavouring a fresh biotechnological perspective for the bioactives secreted by these important and ubiquitous animals.

The interest on cephalotoxins, albeit when they were not termed as such, rose in the late XIX century when Lo Bianco (1888) studied the toxicity of octopus saliva to crabs. These observations showed that after injecting secretions from octopus’ posterior salivary glands directly into crab gills, the target experienced hindered locomotion, progressing rapidly towards complete immobilisation and eventually death (see also Ghiretti, 1960). The active substance that causes this paralysing action was latter purified by Ghiretti (1959) from the posterior salivary glands of Sepia officinalis, identified as a proteinaceous compound and called cephalotoxin. From that time onwards, the composition of salivary secretions of cephalopods gained attention and several constituents have since been isolated and identified, with emphasis on neurotoxins and neuropeptides (Table 1). It is now clear that the complexity of these secretions is reflected in a broad range of biologically active compounds, proteinaceous or not. This includes biological amines, namely tyramine, histamine, p-hydroxyphenyl-ethanol-amine (octopamine), 5-hydroxytryptamine (5-HT or serotonin), noradrenaline or dopamine (Henze, 1913; Bottazzi and Valentini, 1924; Erspamer, 1948; von Euler, 1952; Erspamer and Asero, 1953; Hartman et al., 1960). Several enzymes have also been identified in cephalopod saliva, such as hyaluronidase, chitinase, phospholipase A2, peptidase S1, metalloprotease, carboxypeptidase (Romanini, 1952; Fry et al., 2009; Cornet et al., 2014; Whitelaw et al., 2016), as well as tachykinins (Eledoisin, OctTK-I, and OctTK-II) and CAP domain- (CRISP [Cysteine-rich secretory proteins], Antigen 5 [Ag5], and Pathogenesis-related [PR-1]) -bearing proteins (Erspamer and Anastasi, 1962; Kanda et al., 2003; Fry et al., 2009). However, the number of cephalopod species covered in terms of its toxin identification is still acknowledged to be very low (Cooke et al., 2017). From both Superorders of Class Cephalopoda, Octopodiformes accounts for the highest number of investigated species (about 14 from a total of 300). As for Decapodiformes, i.e., cuttlefishes and squids, not even 2% of all known species have been studied for the characterisation of salivary gland secretions. The number of species for which there is detailed information on cephalotoxins is even smaller. The octopuses Octopus vulgaris, Hapalochlaena maculosa, and Eledone cirrhosa, were the first to have α- and β-cephalotoxin, hapalotoxin, and Eledone toxin identified, respectively (McDonald and Cottrell, 1972; Cariello and Zanetti, 1977; Savage and Howden, 1977). Indeed, Hapalochlaena spp. (the famous blue-ringed octopus) is particularly known for the potency of its neurotoxic agents, which are lethal and devoid of known antidote. Later, Sheumack et al. (1978) was able to identify tetrodotoxin (TTX) in the posterior salivary glands of the octopus Hapalochlaena maculosa as well. This non-peptidic neurotoxin is not, however, exclusive to octopus’ salivary glands since it was found in multiple body parts of the animal, including arms, cephalothorax and abdomen (Yotsu-Yamashita et al., 2007). It must be noted, though, that TTX is a secondary metabolite best-known from pufferfishes (Tetraodontidae) as well as in other marine animals and amphibians, with its synthesis being associated to endosymbiotic bacteria (Lago et al., 2015). Despite the aforementioned studies, information on the bioreactivity and structural properties of cephalotoxins is still very limited. However, cephalotoxins from octopuses and Sepia esculenta have been conclusively identified as functional proteins. The latter species, in particular, yielded, to date, the only complete cephalotoxin amino acid sequence available from a curated record at UniProt, which illustrates how little is known about these substances whose existence is known for more than a century. Toxins from Sepia pharaonis and Sepia prashadi, and from the squid Doryteuthis (Amerigo) pealeii have also been identified as unknown cytotoxic proteins secreted by posterior salivary glands (Kem and Scott, 1980; and purified by Karthik et al. (2015, 2019), which may indicate potential cephalotoxin candidates.

As mentioned earlier, it is believed that the immobilising or paralysing activity of cephalotoxins against invertebrate prey, especially arthropods, can result from the interference with specific ion channels or receptors. On the very least, cephalotoxins could be explored for the development of natural and safer pesticides against arthropods (namely insecticides). Nonetheless, verified effects on murines open excellent prospects for biomedical applications, similarly to conotoxins and TTX, especially for the development of safer and non-addictive painkillers. In fact, Cariello and Zanetti (1977) reported cephalotoxins from O. vulgaris to be able to block glutamate-mediated synaptic transmission, a signalling process involved precisely in pain sensation (see for instance Wozniak et al., 2012). Nonetheless, only few bioactives from the cephalopod salivary glands are indeed under scientific scrutiny for potential applications. Among such studies, research on extracts from Sepia pharaonis and Sepia prashadi posterior salivary glands revealed antimicrobial activity against avian bacterial pathogens and also a potential function as an anti-metastatic agent (Karthik et al., 2015, 2017, 2019). These findings suggest that, adding to neurotoxic effects, biocidal and anti-proliferative properties of cephalotoxins and other components of cephalopod venoms can point to novel antibiotics and anti-cancer drugs, further confirming the rising interest of cephalopod toxins for biotechnology. It must be noted that even TTX, albeit being ubiquitous among many animal taxa (cephalopods included) and one of the best-known and most lethal non-peptidic neural ion channel blockers, has also been considered for the development of analgesic and anaesthetic drugs in advanced cancer patients (Hagen et al., 2007). Indeed, under the trade name Tectin, TTX is already in phase III trials for the treatment of pain resulting from chemotherapy treatments (Newman and Cragg, 2014). To these applications we may add the potential deployment of TTX in the treatment for opioid dependence and management of withdrawal symptoms, with has already started phase IIa clinical trials as Tetrodin (Butler, 2005; Shi et al., 2009; Song et al., 2011). Similarly, the TTX-based drug Tocudin, has also started preclinical studies as a formulation intended for local anaesthesia (Butler, 2005). These findings highlight the high prospects set upon potential applications of marine animal toxins. Proteinaceous toxins such as cephalotoxins and conotoxins may offer advantages over those derived from primary or secondary metabolites, as they can be safer, more easily eliminated (by proteolytic activity) and, very importantly, directly cloned into adequate vectors for heterologous expression. In the latter case, constraints posed by protein secretion and post-translational modifications, such as glycosylation, can be circumvented by choosing eukaryote models, like yeast.

Even though the discovery and description of coleoid venomous secretions dates back to the XIX century, their toxins remain obscure. Cephalotoxins, albeit being consensually found to be the most bioreactive substances in the venom secreted by the posterior salivary gland of these animals (whereas the anterior gland provides the mucin-rich vehicle of delivery), are no exception. Their potent neurotoxic and potential cytotoxic effects, together with the proteinaceous nature, makes them particularly appealing for drug discovery. Such endeavour requires, nonetheless, more solid knowledge on their molecular structure and their interaction with target receptors. With this respect, “omics” approaches, particularly proteomics and transcriptomics, can greatly assist in the filling-in of proteins secreted by the salivary glands of cephalopods by screening multiple peptides and proteins in single runs. These approaches may therefore provide a scaffold to support downstream finer structural details of the substances. The “big picture” must also be complemented with finely tuned toxicity and bioreactivity testing to provide the comprehensive bottom-up perspective needed to devise effective applications.

CG and PC designed the conceptual framework of the manuscript. CG collected information and drafted the manuscript with important input and supervision from PC. Both authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.