Marine Snail Conus Research Articles

Machine Learning Framework for Conotoxin Class and Molecular Target Prediction

Conotoxins are small and highly potent neurotoxic peptides derived from the venom of marine cone snails which have captured the interest of the scientific community due to their pharmacological potential. These toxins display significant sequence and structure diversity, which results in a wide range of specificities for several different ion channels and receptors. Despite the recognized importance of these compounds, our ability to determine their binding targets and toxicities remains a significant challenge. Predicting the target receptors of conotoxins, based solely on their amino acid sequence, remains a challenge due to the intricate relationships between structure, function, target specificity, and the significant conformational heterogeneity observed in conotoxins with the same primary sequence. We have previously demonstrated that the inclusion of post-translational modifications, collisional cross sections values, and other structural features, when added to the standard primary sequence features, improves the prediction accuracy of conotoxins against non-toxic and other toxic peptides across varied datasets and several different commonly used machine learning classifiers. Here, we present the effects of these features on conotoxin class and molecular target predictions, in particular, predicting conotoxins that bind to nicotinic acetylcholine receptors (nAChRs). We also demonstrate the use of the Synthetic Minority Oversampling Technique (SMOTE)-Tomek in balancing the datasets while simultaneously making the different classes more distinct by reducing the number of ambiguous samples which nearly overlap between the classes. In predicting the alpha, mu, and omega conotoxin classes, the SMOTE-Tomek PCA PLR model, using the combination of the SS and P feature sets establishes the best performance with an overall accuracy (OA) of 95.95%, with an average accuracy (AA) of 93.04%, and an f1 score of 0.959. Using this model, we obtained sensitivities of 98.98%, 89.66%, and 90.48% when predicting alpha, mu, and omega conotoxin classes, respectively. Similarly, in predicting conotoxins that bind to nAChRs, the SMOTE-Tomek PCA SVM model, which used the collisional cross sections (CCSs) and the P feature sets, demonstrated the highest performance with 91.3% OA, 91.32% AA, and an f1 score of 0.9131. The sensitivity when predicting conotoxins that bind to nAChRs is 91.46% with a 91.18% sensitivity when predicting conotoxins that do not bind to nAChRs.

The α3β4 nAChR tissue distribution identified by fluorescent α-conotoxin [D11A]LvIA

α3β4, a vital subtype of neuronal nicotinic acetylcholine receptors (nAChRs), is widely distributed in the brain, ganglia, and adrenal glands, associated with addiction and neurological diseases. However, the lack of specific imaging tools for α3β4 nAChRs has hindered the investigation of their tissue distribution and functions. [D11A]LvIA, a peptide derived from marine cone snails, demonstrates high affinity and potency for α3β4 nAChRs, making it a valuable pharmacological tool for studying this receptor subtype. In this study, three fluorescent conjugates of [D11A]LvIA were synthesized using 6-TAMRA-SE (R), Cy3-NHS-ester (Cy3), and BODIPY-FL NHS ester (BDP) dyes. The electrophysiological activities were assessed in Xenopus laevis oocytes by two-electrodes voltage clamp (TEVC). [D11A]LvIA-Cy3 and [D11A]LvIA-BDP show improved selectivity and affinity, with IC50 values of 512.70 nM and 343.50 nM, respectively, and [D11A]LvIA-Cy3 exhibits better stability in cerebrospinal fluid (CSF). Utilizing [D11A]LvIA-Cy3, we successfully visualized the distribution of α3β4 nAChRs in rat trigeminal ganglia, retina, adrenal glands, and various brain regions. This novel fluorescent peptide provides a significant pharmacological tool for the exploration and visualization in-situ distribution of α3β4 nAChRs in different tissues and also assists in clarifying the function.

Conotoxins: emerging analgesics for neck and spinal surgery pain management

Conotoxins, peptides derived from the venom of marine cone snails, have emerged as promising analgesics for managing pain associated with neck and spinal surgery. These toxins target specific neurotransmitter receptors and ion channels in the nervous system, offering an alternative to traditional opioids with potentially fewer side effects. By interacting with receptors such as nicotinic acetylcholine and voltage-gated sodium and calcium channels, conotoxins disrupt pain signal transmission and induce muscle relaxation, providing effective pain relief. Research into conotoxins is ongoing, with the goal of developing novel, safer analgesics that mitigate the risks of opioid addiction. This exploration not only holds promise for surgical pain management but also advances our understanding of venom pharmacology and its therapeutic applications.

Structure-aided function assignment to the transcriptomic conopeptide Am931

Implementation of the next-generation technologies for gene sequencing of venom duct transcriptome has provided a large number of peptide sequences of marine cone snails. Emerging technologies on computational platforms are now rapidly evolving for the accurate predictions of the 3D structure of the polypeptide using the primary sequence. The current report aims to integrate the information derived from these two technologies to develop the concept of structure-aided function assignment of Conus peptides. The proof of the concept was demonstrated using the transcriptomic peptide Am931 of C. amadis. The 3D structure of Am931 was computed using Density Functional Theory (DFT) and the quality of the predicted structure was confirmed using 2D NMR spectroscopy of the corresponding synthetic peptide. The computed structure of Am931 aligns with the active site motif of thioredoxins, possess catalytic disulfide conformation of (+, −)AntiRHHook and selectively modulate the N-terminal Cys3 thiol. These structural features indicate that Am931 may act as a disulfide isomerase and modulate the oxidative folding of conotoxins. Synthetic peptide Am931 provides proof-of-function by exhibiting catalytic activity on the oxidative folding of α-conotoxin ImI and improving the yield of native globular fold. The approach of integration of new technologies in the Conus peptide research may help to accelerate the discovery pipeline of new/improved conotoxin functional.

Prey Shifts Drive Venom Evolution in Cone Snails.

Venom systems are complex traits that have independently emerged multiple times in diverse plant and animal phyla. Within each venomous lineage there typically exists interspecific variation in venom composition where several factors have been proposed as drivers of variation, including phylogeny and diet. Understanding these factors is of broad biological interest and has implications for the development of antivenom therapies and venom-based drug discovery. Because of their high species richness and the presence of several major evolutionary prey shifts, venomous marine cone snails (genus Conus) provide an ideal system to investigate drivers of interspecific venom variation. Here, by analyzing the venom gland expression profiles of ∼3,000 toxin genes from 42 species of cone snail, we elucidate the role of prey-specific selection pressures in shaping venom variation. By analyzing overall venom composition and individual toxin structures, we demonstrate that the shifts from vermivory to piscivory in Conus are complemented by distinct changes in venom composition independent of phylogeny. In vivo injections of venom from piscivorous cone snails in fish further showed a higher potency compared with venom of nonpiscivores demonstrating a selective advantage. Together, our findings provide compelling evidence for the role of prey shifts in directing the venom composition of cone snails and expand our understanding of the mechanisms of venom variation and diversification.

Design, Synthesis, and Structure-Activity Relationships of Novel Peptide Derivatives of the Severe Acute Respiratory Syndrome-Coronavirus-2 Spike-Protein that Potently Inhibit Nicotinic Acetylcholine Receptors.

The spike-protein of SARS-CoV-2 has a distinctive amino-acid sequence (682RRARS686) that forms a cleavage site for the enzyme furin. Strikingly, the structure of the spike-protein loop containing the furin cleavage site bears substantial similarity to neurotoxin peptides found in the venoms of certain snakes and marine cone snails. Leveraging this relationship, we designed and synthesized disulfide-constrained peptides with amino-acid sequences corresponding to the furin cleavage-sites of wild-type (B.1 variant) SARS-CoV-2 or the Alpha, Delta, and Omicron variants. Remarkably, some of these peptides potently inhibited α7 and α9α10 nicotinic acetylcholine receptors (nAChR) with nM affinity and showed SARS-CoV-2 variant and nAChR subtype-dependent potencies. Nuclear magnetic resonance spectroscopy and molecular dynamics were used to rationalize structure-activity relationships between peptides and their cognate receptors. These findings delineate nAChR subtypes that can serve as high-affinity spike-protein targets in tissues central to COVID-19 pathophysiology and identify ligands and target receptors to inform the development of novel SARS-CoV-2 therapeutics.

Fluorescent α-Conotoxin [Q1G, ΔR14]LvIB Identifies the Distribution of α7 Nicotinic Acetylcholine Receptor in the Rat Brain.

α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.

Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Oxidative Folding Catalysts of Conotoxins Derived from the Venom Duct Transcriptome of C. frigidus and C. amadis.

Two novel redox conopeptides with proline residues outside and within the active site disulfide loop were derived from the venom duct transcriptome of the marine cone snails Conus frigidus and Conus amadis. Mature peptides with possible post-translational modification of 4-trans-hydroxylation of proline, namely, Fr874, Fr890[P1O], Fr890[P2O], Fr906, Am1038, and Am1054, have been chemically synthesized and characterized using mass spectrometry. The estimated reduction potential of cysteine disulfides of synthetic peptides varied from -298 to -328 mV, similar to the active site cysteine disulfide motifs of the redox family of proteins. Fr906/Am1054 exhibited pronounced catalytic activity and assisted in improving the yields of natively folded globular form α-conotoxin ImI. Three-dimensional (3D) structures of the redox conopeptides were optimized using computational methods and verified by 2D-ROESY NMR spectroscopy: C. frigidus peptides adopt an N-terminal helical fold and C. amadis peptides adopt distinct structures based on the Phe4-Pro/Hyp5 peptide bond configuration. The shift in the cis-trans configuration of the Phe4-Pro/Hyp5 peptide bond of Am1038/Am1054 was observed between reduced free thiol and oxidized disulfide forms of the optimized peptides. The report confirms the position-specific effect of hydroxyproline on the oxidative folding of conotoxins and sequence diversity of redox conopeptides in the venom duct of cone snails.

Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1–NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure–activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

High-resolution crystal structure of the Mu8.1 conotoxin from Conus mucronatus.

Marine cone snails produce a wealth of peptide toxins (conotoxins) that bind their molecular targets with high selectivity and potency. Therefore, conotoxins constitute valuable biomolecular tools with a variety of biomedical purposes. The Mu8.1 conotoxin from Conus mucronatus is the founding member of the newly identified saposin-like conotoxin class of conotoxins and has been shown to target Cav2.3, a voltage-gated calcium channel. Two crystal structures have recently been determined of Mu8.1 at 2.3 and 2.1 Å resolution. Here, a high-resolution crystal structure of Mu8.1 was determined at 1.67 Å resolution in the high-symmetry space group I4122. The asymmetric unit contained one molecule, with a symmetry-related molecule generating a dimer equivalent to that observed in the two previously determined structures. The high resolution allows a detailed atomic analysis of a water-filled cavity buried at the dimer interface, revealing a tightly coordinated network of waters that shield a lysine residue (Lys55) with a predicted unusually low side-chain pKa value. These findings are discussed in terms of a potential functional role of Lys55 in target interaction.

A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

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, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 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 recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ 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 showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Synthesis of Cystine-Stabilised Dicarba Conotoxin EpI: Ring-Closing Metathesis of Sidechain Deprotected, Sulfide-Rich Sequences.

Recombinant peptide synthesis allows for large-scale production of peptides with therapeutic potential. However, access to dicarba peptidomimetics via sidechain-deprotected sequences becomes challenging with exposed Lewis basicity presented by amine and sulfur-containing residues. Presented here is a combination of strategies which can be used to deactivate coordinative residues and achieve high-yielding Ru-catalyzed ring-closing metathesis. The chemistry is exemplified using α-conotoxin EpI, a native bicyclic disulfide-containing sequence isolated from the marine conesnail Conus episcopatus. Replacement of the loop I disulfide with E/Z-dicarba bridges was achieved with high conversion via solution-phase ring-closing metathesis of the unprotected linear peptide after simple chemoselective oxidation and ion-exchange masking of problematic functionality. Metathesis was also attempted in green solvent choices to further improve the sustainability of dicarba peptide synthesis.

Coordinated adaptations define the ontogenetic shift from worm- to fish-hunting in a venomous cone snail

Marine cone snails have attracted researchers from all disciplines but early life stages have received limited attention due to difficulties accessing or rearing juvenile specimens. Here, we document the culture of Conus magus from eggs through metamorphosis to reveal dramatic shifts in predatory feeding behaviour between post-metamorphic juveniles and adult specimens. Adult C. magus capture fish using a set of paralytic venom peptides combined with a hooked radular tooth used to tether envenomed fish. In contrast, early juveniles feed exclusively on polychaete worms using a unique “sting-and-stalk” foraging behaviour facilitated by short, unbarbed radular teeth and a distinct venom repertoire that induces hypoactivity in prey. Our results demonstrate how coordinated morphological, behavioural and molecular changes facilitate the shift from worm- to fish-hunting in C. magus, and showcase juvenile cone snails as a rich and unexplored source of novel venom peptides for ecological, evolutionary and biodiscovery studies.

Why losing Australia's biodiversity matters for human health: insights from the latest State of the Environment assessment.

Why losing Australia's biodiversity matters for human health: insights from the latest State of the Environment assessment.

Bibliometric Review of the Literature on Cone Snail Peptide Toxins from 2000 to 2022.

The venom of marine cone snails is mainly composed of peptide toxins called conopeptides, among which conotoxins represent those that are disulfide-rich. Publications on conopeptides frequently state that conopeptides attract considerable interest for their potent and selective activity, but there has been no analysis yet that formally quantifies the popularity of the field. We fill this gap here by providing a bibliometric analysis of the literature on cone snail toxins from 2000 to 2022. Our analysis of 3028 research articles and 393 reviews revealed that research in the conopeptide field is indeed prolific, with an average of 130 research articles per year. The data show that the research is typically carried out collaboratively and worldwide, and that discoveries are truly a community-based effort. An analysis of the keywords provided with each article revealed research trends, their evolution over the studied period, and important milestones. The most employed keywords are related to pharmacology and medicinal chemistry. In 2004, the trend in keywords changed, with the pivotal event of that year being the approval by the FDA of the first peptide toxin drug, ziconotide, a conopeptide, for the treatment of intractable pain. The corresponding research article is among the top ten most cited articles in the conopeptide literature. From the time of that article, medicinal chemistry aiming at engineering conopeptides to treat neuropathic pain ramped up, as seen by an increased focus on topological modifications (e.g., cyclization), electrophysiology, and structural biology.

Computational analysis of interactions between AMPA receptor and con-ikot-ikot conotoxin.

Con-ikot-ikot (CII) is a toxin found in the venom of a marine snail Conus striatus, and the only conotoxin known so far to specifically target AMPA receptors (AMPAR) - a subtype of ionotropic glutamate receptors which are important for synaptic transmission and plasticity. It is small in size (∼20 kDa) and has a unique binding site, nestled between the layers of amino-terminal domains (ATDs) and ligand-binding domains (LBDs) of the receptor, which constitute its extracellular portion. Conotoxins and other animal-derived toxins have an established role as important molecular tools in studying various ion channels and some have already been repurposed as therapeutics, therefore we are interested in using CII to study AMPA receptors. In order to gain a better understanding of the interactions between con-ikot-ikot and AMPA receptors, we have performed all-atom molecular dynamics (MD) simulations of a complex of AMPAR extracellular domains (ATDs and LBDs) with glutamate and CII. Network analysis of the resulting MD simulation trajectories, performed using a residue interaction network (RIN)-based tool we are developing, recapitulates previously identified key residue interactions with ligand-binding domains (LBDs) of AMPARs, reveals novel interactions with the LBD layer and suggests interactions between CII and AMPAR amino-terminal domains (ATDs), making CII potentially the only known ligand to interact with AMPAR ATDs. Our new network analysis tool will allow the user to visualise and explore residue interaction networks of single frames, entire trajectories, or sets of trajectories. It will not only be a tool suitable for our needs in investigating the AMPAR/CII complex, but will also act as a general exploratory, hypothesis-generating tool that will be of interest for anyone looking to understand dynamic networks of protein-protein interactions.

Discovery and optimisation of conotoxin Vc1.1 and analogues with analgesic properties

A specimen of the marine cone snail Conus victoriae collected from a beach in Broome, Western Australia, by a group from The University of Melbourne led to the discovery of the α-conotoxin Vc1.1, which was found to have analgesic activity in rodents. The discovery of this venom-derived peptide led to a series of structural, mechanistic and pharmacological studies directed towards the development of a new analgesic for neuropathic pain by groups in Australia and internationally. Solid-phase peptide synthesis played an important role in developing structure–activity relationships. Studies in a rat model of neuropathic pain showed that a cyclic analogue of the peptide, cVc1.1, had comparable analgesic activity with that of gabapentin, one of the foremost clinically used drugs for neuropathic pain, with cVc1.1 delivered orally at a 120-fold lower dose than gabapentin. Originally, Vc1.1 was believed to act primarily through nicotinic acetylcholine receptors, but evidence for a mechanism mediated through γ-aminobutyric acid B (GABAB) receptors later emerged. Efforts to optimise the binding and pharmacological properties of analogues of Vc1.1 revealed that the affinity towards either receptor can be modulated by sequence mutations, disulfide bond modifications and backbone cyclisation. This Account describes the discovery, structure, chemistry and pharmacology of Vc1.1, with a focus on studies carried out in Australian laboratories.

High conopeptide diversity in Conus striatus: Revealed by integration of two transcriptome sequencing platforms

Marine cone snail venoms represent a vast library of bioactive peptides with proven potential as research tools, drug leads, and therapeutics. In this study, a transcriptome library of four different organs, namely radular sheath, venom duct, venom gland, and salivary gland, from piscivorous Conus striatus was constructed and sequenced using both Illumina next-generation sequencing (NGS) and PacBio third-generation sequencing (TGS) technologies. A total of 428 conotoxin precursor peptides were retrieved from these transcriptome data, of which 413 conotoxin sequences assigned to 13 gene superfamilies, and 15 conotoxin sequences were classified as unassigned families. It is worth noting that there were significant differences in the diversity of conotoxins identified from the NGS and TGS data: 82 conotoxins were identified from the NGS datasets while 366 conotoxins from the TGS datasets. Interestingly, we found point mutations in the signal peptide sequences of some conotoxins with the same mature sequence. Therefore, TGS broke the traditional view of the conservation of conotoxin signal peptides and the variability of mature peptides obtained by NGS technology. These results shed light on the integrated NGS and TGS technologies to mine diverse conotoxins in Conus species, which will greatly contribute to the discovery of novel conotoxins and the development of new marine drugs.