Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Hematophagy arose independently multiple times during metazoan evolution, with several lineages of vampire animals particularly diversified in invertebrates. However, the biochemistry of hematophagy has been studied in a few species of direct medical interest and is still underdeveloped in most invertebrates, as in general is the study of venom toxins. In cone snails, leeches, arthropods and snakes, the strong target specificity of venom toxins uniquely aligns them to industrial and academic pursuits (pharmacological applications, pest control etc.) and provides a biochemical tool for studying biological activities including cell signalling and immunological response. Neogastropod snails (cones, oyster drills etc.) are carnivorous and include active predators, scavengers, grazers on sessile invertebrates and hematophagous parasites; most of them use venoms to efficiently feed. It has been hypothesized that trophic innovations were the main drivers of rapid radiation of Neogastropoda in the late Cretaceous. We present here the first molecular characterization of the alimentary secretion of a non-conoidean neogastropod, Colubraria reticulata. Colubrariids successfully feed on the blood of fishes, throughout the secretion into the host of a complex mixture of anaesthetics and anticoagulants. We used a NGS RNA-Seq approach, integrated with differential expression analyses and custom searches for putative secreted feeding-related proteins, to describe in detail the salivary and mid-oesophageal transcriptomes of this Mediterranean vampire snail, with functional and evolutionary insights on major families of bioactive molecules. A remarkably low level of overlap was observed between the gene expression in the two target tissues, which also contained a high percentage of putatively secreted proteins when compared to the whole body. At least 12 families of feeding-related proteins were identified, including: 1) anaesthetics, such as ShK Toxin-containing proteins and turripeptides (ion-channel blockers), Cysteine-rich secretory proteins (CRISPs), Adenosine Deaminase (ADA); 2) inhibitors of primary haemostasis, such as novel vWFA domain-containing proteins, the Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5) and the wasp Antigen-5; 3) anticoagulants, such as TFPI-like multiple Kunitz-type protease inhibitors, Peptidases S1 (PS1), CAP/ShKT domain-containing proteins, Astacin metalloproteases and Astacin/ShKT domain-containing proteins; 4) additional proteins, such the Angiotensin-Converting Enzyme (ACE: vasopressive) and the cytolytic Porins. Colubraria feeding physiology seems to involve inhibitors of both primary and secondary haemostasis, anaesthetics, a vasoconstrictive enzyme to reduce feeding time and tissue-degrading proteins such as Porins and Astacins. The complexity of Colubraria venomous cocktail and the divergence from the arsenal of the few neogastropods studied to date (mostly conoideans) suggest that biochemical diversification of neogastropods might be largely underestimated and worth of extensive investigation.

Many marine mollusks attain or increase their predatory efficiency using complex chemical secretions, which are often produced and delivered through specialized anatomical structures of the foregut. The secretions produced in venom glands of Conus snails and allies have been extensively studied, revealing an amazing chemical diversity of small, highly constrained neuropeptides, whose characterization led to significant pharmacological developments. Conversely, salivary glands, the other main secretory structures of molluscan foregut, have been neglected despite their shared occurrence in the two lineages including predatory members: Gastropoda and Cephalopoda. Over the last few years, the interest for the chemistry of salivary mixtures increased based on their potential biomedical applications. Recent investigation with -omics technologies are complementing the classical biochemical descriptions, that date back to the 1950s, highlighting the high level of diversification of salivary secretions in predatory mollusks, and suggesting they can be regarded as a pharmaceutical cornucopia. As with other animal venoms, some of the salivary toxins are reported to target, for example, sodium and/or potassium ion channels or receptors and transporters for neurotransmitters such as glutamate, serotonin, neurotensin and noradrenaline, thus manipulating the neuromuscular system of the preys. Other bioactive components possess anticoagulant, anesthetic and hypotensive activities. Here we overview available knowledge on the salivary glands of key predatory molluscan taxa, gastropods and cephalopods, summarizing their anatomical, physiological and biochemical complexity in order to facilitate future comparative studies on main evolutionary trends and functional convergence in the acquisition of successful predatory strategies. As with other animal venoms, some of the salivary toxins are reported to target, for example, sodium and/or potassium ion channels or receptors and transporters for neurotransmitters such as glutamate, serotonin, neurotensin and noradrenaline, thus manipulating the neuromuscular system of the preys. Other bioactive components possess anticoagulant, anaesthetic and hypotensive activities. Here we review available knowledge for different species belonging to key predatory Molluscan taxa, including gastropods and cephalopods, with the aims of highlighting main evolutionary trends both at the anatomical and at the physiological and biochemical level, and understanding the role of functional convergence in the definition of successful predatory strategies among different taxa.

The diversity of venomous organisms and the toxins they produce have been increasingly investigated, but taxonomic bias remains important. Neogastropods, a group of marine predators representing almost 22% of the known gastropod diversity, evolved a wide range of feeding strategies, including the production of toxins to subdue their preys. However, whether the diversity of these compounds is at the origin of the hyperdiversification of the group and how genome evolution may correlate with both the compounds and species diversities remain understudied. Among the available gastropods genomes, only eight, with uneven quality assemblies, belong to neogastropods. Here, we generated chromosome-level assemblies of two species belonging to the Tonnoidea and Muricoidea superfamilies (Monoplex corrugatus and Stramonita haemastoma). The two obtained high-quality genomes had 3 and 2.2 Gb, respectively, and 92–89% of the total assembly conformed 35 pseudochromosomes in each species. Through the analysis of syntenic blocks, Hox gene cluster duplication, and synonymous substitutions distribution pattern, we inferred the occurrence of a whole genome duplication event in both genomes. As these species are known to release venom, toxins were annotated in both genomes, but few of them were found in homologous chromosomes. A comparison of the expression of ohnolog genes (using transcriptomes from osphradium and salivary glands in S. haemastoma), where both copies were differentially expressed, showed that most of them had similar expression profiles. The high quality of these genomes makes them valuable reference in their respective taxa, facilitating the identification of genome-level processes at the origin of their evolutionary success.