Maren Watkins

New genes and gene functions are key drivers of evolutionary innovation. Venomous animals, such as cone snails, provide striking examples of gene innovation, yet the mechanisms by which toxins arise remain poorly understood. Using the Conus textile genome, we uncover how neuropeptide genes were recruited into the venom and neofunctionalized as doppelgänger toxins. We identify over 20 independent recruitment events that evolved dynamically across the Conus lineage. Rather than arising from ohnologs of a whole-genome duplication event ∼200 mya, these toxins evolved through diverse mechanisms, including exon shuffling, alternative splicing, and ectopic recombination, often facilitated by lineage-specific transposable elements. Our findings reveal a dynamic interplay between genome architecture and molecular innovation, offering broad insight into the evolution of complex gene repertoires in venoms and beyond.

Due to their remarkable diversity and rapid evolution, conotoxins—peptide toxins from predatory marine cone snails—provide a powerful system for exploring how gene diversification may contribute to the development of lineage-specific adaptations. We previously demonstrated that 2-loop Tau conotoxins represent an evolutionary innovation associated with mollusk-hunting behaviors in cone snails. Here, we investigate the evolutionary history of these toxins as a model to understand the mechanism of ancestral gene neofunctionalization, which may have contributed to the emergence of mollusk-hunting in cone snails. Using ancestral sequence reconstruction, we present a model in which ancestral T-superfamily conotoxins neofunctionalized into the 2-loop Tau conotoxins. Predicted ancestral sequences reveal an intermediate structure between the classic T-superfamily conotoxins and the derived 2-loop Tau forms. Notably, these ancestral intermediates acquired a new cysteine scaffold that facilitated a structural transition from a globular to a ribbon fold. This conformational shift was followed by sequence-level changes that presumably enhanced activity against molecular targets in mollusks. We propose that the emergence of 2-loop Tau conotoxins may have been one factor that contributed to the emergence of molluscivory, providing insight into how gene innovation may underlie ecological diversification.

Conotoxins, peptides found in cone snail venoms, selectively target ion channels and receptors to incapacitate prey. α‐Conotoxins are extensively investigated for their potent modulation of nicotinic acetylcholine receptors (nAChRs). This study describes the discovery and characterization of RoIA, a novel α‐conotoxin from Conus rolani . RoIA belongs to the α4/4 conotoxin class and features an atypical N‐terminal elongation of 18 amino acids. The biological activity of RoIA is assessed through both in vivo and in vitro assays using the three potential folding isoforms (globular, ribbon, and bead) of the full‐length and truncated (lacking the N‐terminal elongation) analogs. The full‐length RoIA analog exhibits delayed but potent paralytic activity when administered to mice and fish; the ribbon isoform shows the highest potency. Notably, only the ribbon isoform of the truncated peptide is active on heterologously expressed muscle nAChRs, suggesting that the N‐terminal elongation may be released in vivo or form interactions that are not recapitulated in vitro. This discovery challenges the prevailing understanding that native α‐conotoxins adopt a globular conformation and illustrates that Conus can explore novel chemical spaces using an alternative disulfide bond connectivity. This research enhances our knowledge of the complex mechanisms by which toxins manifest their physiological effects.

Alan Kohn showed that two cone-snail species—Conus striatus Linnaeus, 1758, and Conus catus Hwass, 1792—hunt fish as their primary prey. In the seven decades since then, it has been established that these two species belong to the subgenus Pionoconus Mörch, 1852, a well-defined lineage of Conus Linnaeus, 1758, and one of eight piscivorous cone-snail clades. In this review, an integrated multidisciplinary framework for the biology of Pionoconus is presented, based on the numerous research papers published since Kohn's seminal discovery. The molecular biology, phylogeny, biogeography, life history, and prey-capture strategy of Pionoconus are among the topics reviewed, along with more specialized subjects, e.g., human envenomation, culinary recipes, shell-collecting history, and biomedical applications. These illustrate the continuing impact of Kohn's scientific contributions.

Mollusk-hunting (molluscivorous) cone snails belong to a monophyletic group in Conus, a genus of venomous marine snails. The molluscivorous lineage evolved from ancestral worm-hunting (vermivorous) snails ∼18 million years ago. To enable the shift to a molluscivorous lifestyle, molluscivorous cone snails must solve biological problems encountered when hunting other gastropods, namely: 1) preventing prey escape and 2) overcoming the formidable defense of the prey in the form of the molluscan shell, a problem unique to molluscivorous Conus. Here we show that χ-conotoxins, peptides exclusively expressed in the venoms of molluscivorous Conus, provide solutions to the above problems. Injecting χ-conotoxins into the gastropod mollusk Aplysia californica results in impaired locomotion and uncoordinated hyperactivity. Impaired locomotion impedes escape and a hyperactive snail will likely emerge from its shell, negating the protection the shell provides. Thus, χ-conotoxins are an evolutionary innovation that accompanied the emergence of molluscivory in Conus and provide solutions to problems posed by hunting other snails.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3β4, α6/α3β4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3β4 and α6/α3β4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and β2 subunits (α7β2‐nAChR subtype). Basal forebrain cholinergic neurons express α7β2‐nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid‐β associated with early Alzheimer's disease. Additional work indicates that α7β2‐nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7β2‐nAChR from the closely related and more widespread homomeric α7‐only‐nAChR subtype. Functional screening using two‐electrode voltage‐clamp electrophysiology identified a family of α7β2‐nAChR‐selective analogs of α‐conotoxin PnIC (α‐CtxPnIC). A combined electrophysiology, functional kinetics, site‐directed mutagenesis, and molecular dynamics approach was used to further characterize the α7β2‐nAChR selectivity and site of action of these α‐CtxPnIC analogs. We determined that α7β2‐nAChR selectivity of α‐CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist‐binding site shared between α7β2‐ and α7‐only‐nAChR. As numerous previously identified α‐Ctx ligands are competitive antagonists of orthosteric agonist‐binding sites, this study profoundly expands the scope of use of α‐Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α‐CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7β2‐nAChR and detailed investigations of their physiological roles.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and two additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca ²⁺ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K ⁺ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.

Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.

The venomous marine snails are conventionally divided into three groups, the cone snails (family Conidae), the auger snails (family Terebridae) and the turrids (formerly all assigned to a single family, Turridae). In this study, a library of venom peptides from species conventionally assigned to the genus Turris was correlated to a phylogenetic analysis. Nucleotide sequences of multiple genes from transcriptomes were used to assess the phylogenetic relationships across a diverse set of species. The resulting tree shows that as conventionally defined, the conoidean genus Turris, is polyphyletic. We describe a new genus, Purpuraturris gen. nov., that comprises the outlier species. In addition to morphological distinctions, molecular data reveal that this group is divergent from Turris sensu stricto. The correlation between phylogenetic information and a family of peptide sequences was used to highlight those peptides mostly likely to be unique and intimately associated with biological diversity. The plethora of peptide sequences available requires two prioritization decisions: which subset of peptides to initially characterize, and after these are characterized, which to comprehensively investigate for potential biomedical applications such as drug developments.Life Science Identifiers: urn:lsid:zoobank.org; pub: 60D46561-28F0-4C39-BAC4-66DC8B4EAEA4