Search

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii . Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC 50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC 50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.

Within the Conidae family, the piscivorous Conus species have been a hotspot target for drug discovery. Here, we assess the relevance of Conus and their other feeding habits, and thus under distinctive evolutionary constraints, to highlight the potential of neglected molluscivorous and vermivorous species in biomedical research and pharmaceutical industry. By singling out the areas with inadequate Conus disquisition, such as the Tamil Nadu Coast and the Andaman Islands, research resources can be expanded and better protected through awareness. In this study, 728 Conus species and 190 species from three other genera (1 from Californiconus , 159 from Conasprella and 30 from Profundiconus ) in the Conidae family are assessed. The phylogenetic relationships of the Conidae species are determined and their known feeding habits superimposed. The worm-hunting species appeared first, and later the mollusc- and fish-hunting species were derived independently in the Neogene period (around 23 million years ago). Interestingly, many Conus species in the warm and shallow waters become polyphagous, allowing them to hunt both fish and worms, given the opportunities. Such newly gained trait is multi originated. This is controversial, given the traditional idea that most Conus species are specialized to hunt certain prey categories. However, it shows the functional complexity and great potential of conopeptides from some worm-eating species. Pharmaceutical attempts and relevant omics data have been differentially obtained. Indeed, data from the fish-hunting species receive strong preference over the worm-hunting ones. Expectedly, conopeptides from the fish-hunting species are believed to include the most potential candidates for biomedical research. Our work revisits major findings throughout the Conus evolution and emphasizes the importance of increasing omics surveys complemented with further behavior observation studies. Hence, we claim that Conus species and their feeding habits are equally important, highlighting many places left for Conus exploration worldwide. We also discuss the Conotoxin drug discovery potentials and the urgency of protecting the bioresources of Conus species. In particular, some vermivorous species have demonstrated great potential in malaria therapy, while other conotoxins from several worm- and mollusc-eating species exhibited explicit correlation with SARS-CoV-2. Reclaiming idle data with new perspectives could also promote interdisciplinary studies in both virological and toxicological fields.

The short disulfide-rich α-conotoxins derived from the venom of Conus snails comprise a conserved C I C II (m)C III (n)C IV cysteine framework (m and n, number of amino acids) and the majority antagonize nicotinic acetylcholine receptors (nAChRs). Depending on disulfide connectivity, α-conotoxins can exist as either globular (C I -C III , C II -C IV ), ribbon (C I -C IV , C II -C III ) or bead (C I -C II , C III -C IV ) isomers. In the present study, C. geographus α-conotoxins GI, GIB, G1.5 and G1.9 were chemically synthesized as globular and ribbon isomers and their activity investigated at human nAChRs expressed in Xenopus oocytes using the two-electrode voltage clamp recording technique. Both the globular and ribbon isomers of the 3/5 (m/n) α-conotoxins GI and GIB selectively inhibit heterologous human muscle-type α1β1δε nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3β2, α7 and α9α10 nAChRs. In contrast, globular and ribbon isomers of G1.9, a novel C-terminal elongated 4/8 α-conotoxin exhibited no activity at the human nAChR subtypes studied. This study reinforces earlier observations that 3/5 α-conotoxins selectively target the muscle nAChR subtypes, although interestingly, GIB is also active at α7 and α9 α10 nAChRs. The 4/7 α-conotoxins target human neuronal nAChR subtypes whereas the pharmacology of the 4/8 α-conotoxin remains unknown., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The venom duct origins of predatory and defensive venoms has not been studied for hook-and-line fish hunting cone snails despite the pharmacological importance of their venoms. To better understand the biochemistry and evolution of injected predatory and defensive venoms, we compared distal, central and proximal venom duct sections across three specimens of C. striatus (Pionoconus) using proteomic and transcriptomic approaches. A total of 370 conotoxin precursors were identified from the whole venom duct transcriptome. Milked defensive venom was enriched with a potent cocktail of proximally expressed inhibitory α-, ω- and μ-conotoxins compared to milked predatory venom. In contrast, excitatory κA-conotoxins dominated both the predatory and defensive venoms despite their distal expression, suggesting this class of conotoxin can be selectively expressed from the same duct segment in response to either a predatory or defensive stimuli. Given the high abundance of κA-conotoxins in the Pionoconus clade, we hypothesise that the κA-conotoxins have evolved through adaptive evolution following their repurposing from ancestral inhibitory A superfamily conotoxins to facilitate the dietary shift to fish hunting and species radiation in this clade.

Marine cone snails belonging to the Conidae family make use of neuroactive peptides in their venom to capture prey. Here we report the proteome profile of the venom duct of Conus eburneus , a cone snail belonging to the Tesseliconus clade. Through tandem mass spectrometry and database searching against the C. eburneus transcriptome and the ConoServer database, we identified 24 unique conopeptide sequences in the venom duct. The majority of these peptides belong to the T and M gene superfamilies and are disulfide-bonded, with cysteine frameworks V, XIV, VI/VII, and III being the most abundant. All seven of the Cys-free peptides are conomarphin variants belonging to the M superfamily that eluted out as dominant peaks in the chromatogram. These conomarphins vary not only in amino acid residues in select positions along the backbone but also have one or more post-translational modifications (PTMs) such as proline hydroxylation, C-term amidation, and γ-carboxylation of glutamic acid. Using molecular dynamics simulations, the conomarphin variants were predicted to predominantly have hairpin-like or elongated structures in acidic pH. These two structures were found to have significant differences in electrostatic properties and the inclusion of PTMs seems to complement this disparity. The presence of polar PTMs (hydroxyproline and γ-carboxyglutamic acid) also appear to stabilize hydrogen bond networks in these conformations. Furthermore, these predicted structures are pH sensitive, becoming more spherical and compact at higher pH. The subtle conformational variations observed here might play an important role in the selection and binding of the peptides to their molecular targets.

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC 50  = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α 1 -adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

Species interactions are fundamental ecological forces that can have significant impacts on the evolutionary trajectories of species. Nonetheless, the contribution of predator-prey interactions to genetic and phenotypic divergence remains largely unknown. Predatory marine snails of the family Conidae exhibit specializations for different prey items and intraspecific variation in prey utilization patterns at geographic scales. Because cone snails utilize venom to capture prey and venom peptides are direct gene products, it is feasible to examine the evolution of genes associated with changes in resource utilization. Here, we compared feeding ecologies and venom duct transcriptomes of individuals from three populations of Conus miliaris , a species that exhibits geographic variation in prey utilization and dietary breadth, in order to determine the extent to which dietary differences are correlated with differences in venom composition, and if expanded niche breadth is associated with increased variation in venom composition. While populations showed little to no overlap in resource utilization, taxonomic richness of prey was greatest at Easter Island. Changes in dietary breadth were associated with differences in expression patterns and increased genetic differentiation of toxin-related genes. The Easter Island population also exhibited greater diversity of toxin-related transcripts, but did not show increased variance in expression of these transcripts. These results imply that differences in dietary breadth contribute more to the structural and regulatory differentiation of venoms than differences in diet.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis . Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus , and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.

A diverse range of predatory marine gastropods produce toxins, yet most of these molecules remain uncharacterized. Conus species have received the most attention from researchers, leading to several conopeptides reaching clinical trials. This review aims to summarize what is known about bioactive compounds isolated from species of neglected marine gastropods, especially in the Turridae, Terebridae, Babyloniidae, Muricidae, Buccinidae, Colubrariidae, Nassariidae, Cassidae, and Ranellidae families. Multiple species have been reported to contain bioactive compounds with potential toxic activity, but most of these compounds have not been characterized or even clearly identified. The bioactive properties and potential applications of echotoxins and related porins from the Ranellidae family are discussed in more detail. Finally, the review concludes with a call for research on understudied species., Competing Interests: The authors declare no conflicts of interest.

Cone snail venoms are considered a treasure trove of bioactive peptides. Despite over 800 species of cone snails being known, each producing over 1000 venom peptides, only about 150 unique venom peptides are structurally and functionally characterized. To overcome the limitations of the traditional low-throughput bio-discovery approaches, multi-omics systems approaches have been introduced to accelerate venom peptide discovery and characterisation. This "venomic" approach is starting to unravel the full complexity of cone snail venoms and to provide new insights into their biology and evolution. The main challenge for venomics is the effective integration of transcriptomics, proteomics, and pharmacological data and the efficient analysis of big datasets. Novel database search tools and visualisation techniques are now being introduced that facilitate data exploration, with ongoing advances in related omics fields being expected to further enhance venomics studies. Despite these challenges and future opportunities, cone snail venomics has already exponentially expanded the number of novel venom peptide sequences identified from the species investigated, although most novel conotoxins remain to be pharmacologically characterised. Therefore, efficient high-throughput peptide production systems and/or banks of miniaturized discovery assays are required to overcome this bottleneck and thus enhance cone snail venom bioprospecting and accelerate the identification of novel drug leads., Competing Interests: The authors declare no conflict of interest.

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

From a biological perspective, a natural product can be defined as a compound evolved by an organism for chemical interactions with another organism including prey, predator, competitor, pathogen, symbiont or host. Natural products hold tremendous potential as drug leads and have been extensively studied by chemists and biochemists in the pharmaceutical industry. However, the biological purpose for which a natural product evolved is rarely addressed. By focusing on a well-studied group of natural products-venom components from predatory marine cone snails-this review provides a rationale for why a better understanding of the evolution, biology and biochemistry of natural products will facilitate both neuroscience and the potential for drug leads. The larger goal is to establish a new sub-discipline in the broader field of neuroethology that we refer to as "Chemical Neuroethology", linking the substantial work carried out by chemists on natural products with accelerating advances in neuroethology.

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.

Ocean acidification poses a range of threats to marine invertebrates; however, the emerging and likely widespread effects of rising carbon dioxide (CO 2 ) levels on marine invertebrate behaviour are still little understood. Here, we show that ocean acidification alters and impairs key ecological behaviours of the predatory cone snail Conus marmoreus Projected near-future seawater CO 2 levels (975 µatm) increased activity in this coral reef molluscivore more than threefold (from less than 4 to more than 12 mm min -1 ) and decreased the time spent buried to less than one-third when compared with the present-day control conditions (390 µatm). Despite increasing activity, elevated CO 2 reduced predation rate during predator-prey interactions with control-treated humpbacked conch, Gibberulus gibberulus gibbosus; 60% of control predators successfully captured and consumed their prey, compared with only 10% of elevated CO 2 predators. The alteration of key ecological behaviours of predatory invertebrates by near-future ocean acidification could have potentially far-reaching implications for predator-prey interactions and trophic dynamics in marine ecosystems. Combined evidence that the behaviours of both species in this predator-prey relationship are altered by elevated CO 2 suggests food web interactions and ecosystem structure will become increasingly difficult to predict as ocean acidification advances over coming decades., (© 2017 The Author(s).)

The discovery of insulin and its use for the treatment of diabetes is undoubtedly one of the true successes of modern medicine. Injectable insulin would prove the first effective treatment for a previously incurable and usually fatal disease. Soon after however, the powerful effects of insulin overdose would be reported, and subsequently exploited for dubious medical and sometimes nefarious purposes. In this article we describe the discovery that certain venomous marine snails of the genus Conus also exploit the powerful effects of insulin overdose, employing it as a weapon for prey capture., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Venomous organisms serve as wonderful systems to study the evolution and expression of genes that are directly associated with prey capture. To evaluate the relationship between venom gene expression and prey utilization, we examined these features among individuals of different ages of the venomous, worm-eating marine snail Conus ebraeus. We determined expression levels of six genes that encode venom components, used a DNA-based approach to evaluate the identity of prey items, and compared patterns of venom gene expression and dietary specialization., Results: C. ebraeus exhibits two major shifts in diet with age-an initial transition from a relatively broad dietary breadth to a narrower one and then a return to a broader diet. Venom gene expression patterns also change with growth. All six venom genes are up-regulated in small individuals, down-regulated in medium-sized individuals, and then either up-regulated or continued to be down-regulated in members of the largest size class. Venom gene expression is not significantly different among individuals consuming different types of prey, but instead is coupled and slightly delayed with shifts in prey diversity., Conclusion: These results imply that changes in gene expression contribute to intraspecific variation of venom composition and that gene expression patterns respond to changes in the diversity of food resources during different growth stages.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.

The venomous fish-hunting cone snails (Conus) comprise eight distinct lineages evolved from ancestors that preyed on worms. In this article, we attempt to reconstruct events resulting in this shift in food resource by closely examining patterns of behavior, biochemical agents (toxins) that facilitate prey capture and the combinations of toxins present in extant species. The first sections introduce three different hunting behaviors associated with piscivory: 'taser-and-tether', 'net-engulfment' and 'strike-and-stalk'. The first two fish-hunting behaviors are clearly associated with distinct groups of venom components, called cabals, which act in concert to modify the behavior of prey in a specific manner. Derived fish-hunting behavior clearly also correlates with physical features of the radular tooth, the device that injects these biochemical components. Mapping behavior, biochemical components and radular tooth features onto phylogenetic trees shows that fish-hunting behavior emerged at least twice during evolution. The system presented here may be one of the best examples where diversity in structure, physiology and molecular features were initially driven by particular pathways selected through behavior., (© 2015 S. Karger AG, Basel.)

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary "smoking gun" that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide's putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.

The International Union for Conservation of Nature Red List of Endangered Species employs a robust, standardized approach to assess extinction threat focussed on taxa approaching an end-point in population decline. Used alone, we argue this enforces a reactive approach to conservation. Species not assessed as threatened but which occur predominantly in areas with high levels of anthropogenic impact may require proactive conservation management to prevent loss. We matched distribution and bathymetric range data from the global Red List assessment of 632 species of marine cone snails with human impacts and projected ocean thermal stress and aragonite saturation (a proxy for ocean acidification). Our results show 67 species categorized as 'Least Concern' have 70% or more of their occupancy in places subject to high and very high levels of human impact with 18 highly restricted species (range <100 km(2)) living exclusively in such places. Using a range-rarity scoring method we identified where clusters of endemic species are subject to all three stressors: high human impact, declining aragonite saturation levels and elevated thermal stress. Our approach reinforces Red List threatened status, highlights candidate species for reassessment, contributes important evidential data to minimize data deficiency and identifies regions and species for proactive conservation., (© 2014 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure-function relationship data.

Marine molluscs represent an estimated 23% of all extant marine taxa, but research into their conservation status has so far failed to reflect this importance, with minimal inclusion on the authoritative Red List of the International Union for the Conservation of Nature (IUCN). We assessed the status of all 632 valid species of the tropical marine gastropod mollusc, Conus (cone snails), using Red List standards and procedures to lay the groundwork for future decadal monitoring, one of the first fully comprehensive global assessments of a marine taxon. Three-quarters (75.6%) of species were not currently considered at risk of extinction owing to their wide distribution and perceived abundance. However, 6.5% were considered threatened with extinction with a further 4.1% near threatened. Data deficiency prevented 13.8% of species from being categorised although they also possess characteristics that signal concern. Where hotspots of endemism occur, most notably in the Eastern Atlantic, 42.9% of the 98 species from that biogeographical region were classified as threatened or near threatened with extinction. All 14 species included in the highest categories of Critically Endangered and Endangered are endemic to either Cape Verde or Senegal, with each of the three Critically Endangered species restricted to single islands in Cape Verde. Threats to all these species are driven by habitat loss and anthropogenic disturbance, in particular from urban pollution, tourism and coastal development. Our findings show that levels of extinction risk to which cone snails are exposed are of a similar magnitude to those seen in many fully assessed terrestrial taxa. The widely held view that marine species are less at risk is not upheld.

Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con) Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR). We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg(2+). Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg(2+). Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca(2+) (iCa(2+)) influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A(-/-) and GluN2B(-/-) mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A(-/-)-derived mouse neurons, as compared to those isolated from GluN2B(-/-)-mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser(133), suggesting that the peptides modulated iCa(2+) entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa(2+) influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.

The venom of cone snails (ssp. Conus), a genus of predatory mollusks, is a vast source of bioactive peptides. Conus venom expression is complex, and venom composition can vary considerably depending upon the method of extraction and the species of cone snail in question. The injected venom from Conus ermineus, the only fish-hunting cone snail species that inhabits the Atlantic Ocean, was characterized using nanoNMR spectroscopy, MALDI-TOF mass spectrometry, RP-HPLC and nanoLC-ESI-MS. These methods allowed us to evaluate the variability of the venom within this species. Single specimens of C. ermineus show unchanged injected venom mass spectra and HPLC profiles over time. However, there was significant variability of the injected venom composition from specimen to specimen, in spite of their common biogeographic origin. Using nanoLC-ESI-MS, we determined that over 800 unique conopeptides are expressed by this reduced set of C. ermineus specimens. This number is considerably larger than previous estimates of the molecular repertoire available to cone snails to immobilize prey. These results support the idea of the existence of a complex regulatory mechanism to express specific venom peptides for injection into prey. These intraspecies differences can be a result of a combination of genetic and environmental factors. The differential expression of venom components represents a neurochemical paradigm that warrants further investigation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Most species within the genus Conus are considered to be specialists in their consumption of prey, typically feeding on molluscs, vermiform invertebrates or fish, and employ peptide toxins to immobilize prey. Conus californicus Hinds 1844 atypically utilizes a wide range of food sources from all three groups. Using DNA- and protein-based methods, we analyzed the molecular diversity of C. californicus toxins and detected a correspondingly large number of conotoxin types. We identified cDNAs corresponding to seven known cysteine-frameworks containing over 40 individual inferred peptides. Additionally, we found a new framework (22) with six predicted peptide examples, along with two forms of a new peptide type of unusual length. Analysis of leader sequences allowed assignment to known superfamilies in only half of the cases, and several of these showed a framework that was not in congruence with the identified superfamily. Mass spectrometric examination of chromatographic fractions from whole venom served to identify peptides corresponding to a number of cDNAs, in several cases differing in their degree of posttranslational modification. This suggests differential or incomplete biochemical processing of these peptides. In general, it is difficult to fit conotoxins from C. californicus into established toxin classification schemes. We hypothesize that the novel structural modifications of individual peptides and their encoding genes reflect evolutionary adaptation to prey species of an unusually wide range as well as the large phylogenetic distance between C. californicus and Indo-Pacific species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Conus tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) α3β2, α4β2, and α7, except for C. coronatus venom that blocked the α4β2 and α7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the α-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

With the advent of highly sensitive mass spectrometry techniques, the minute amount of various secretions produced by living animals can be studied to a level of details never attained before. In this study, we used LC-ESI-MS to analyse the injected venom of an indo-pacific piscivorous cone snail, Conus consors. While long-term follow up of several captive specimens have revealed a typical "venom fingerprint" for this species, dramatic variations were also observed. In the most extreme case, a single cone snail unexpectedly produced two very distinct venom profiles containing completely different sets of peptides with no overlap of detected masses. Surprisingly, there was no correlation between the peptides produced in the venom duct and those obtained after milking live cone snails, implying yet unknown mechanisms of selection and regulation. Our study defines the notion of intraspecimen variation and demonstrates how this phenomenon contributes to the overall venom diversity.

Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.

Cone snails use an extensile, tubular proboscis as a conduit to deliver a potent cocktail of bioactive venom peptides into their prey. Previous studies have focused mainly on understanding the venom's role in prey capture but successful prey capture requires both rapid physiological and biomechanical mechanisms. Conus catus, a fish-hunting species, uses a high-speed hydraulic mechanism to inject its hollow, spear-like radular tooth into prey. We take an integrated approach to investigating the biomechanics of this process by coupling kinematic studies with morphological analyses. Taking advantage of the opaque venom and translucent proboscis of a mollusc-hunting juvenile cone snail, Conus pennaceus, we have determined that a high-speed prey capture mechanism is not unique to cone species that hunt fish prey. Two morphological structures were found to play crucial roles in this process. A constriction of the lumen near the tip of the proboscis, composed of tall epithelial cells densely packed with microfilaments, impedes forward movement of the radular tooth prior to its propulsion. Proximal to the constriction, a muscular sphincter was found to regulate venom flow and pressurization in the proboscis. In C. pennaceus, the rapid appearance and flushing of venom within the proboscis during prey capture suggests a mechanism involving the delivery of a discrete quantity of venom. The interplay between these elements provides a unique and effective biomechanical injection system for the fast-acting cone snail venom peptides.

Anomalous mitochondrial and nuclear gene sequences in individuals of the widely distributed tropical marine gastropod Conus ebraeus that were not distinguishable by shell shape and color pattern characters suggested the presence of a second, cryptic species. We tested this hypothesis by genetic, morphological, and ecological comparisons of additional individuals from the site in Okinawa where the two forms co-occurred. Radular tooth size and shape, prey type in nature, and microhabitats utilized differed markedly between the two forms. Adults with typical C. ebraeus DNA and radular teeth preyed primarily on errant polychaetes (Eunicidae); those with anomalous DNA and teeth ate mainly sedentary capitellids. Juveniles (shell length <13 mm) had more similar teeth and ate primarily syllids. Radular teeth of the anomalous form agreed with those of Conus judaeus, distinguished from C. ebraeus by Rudolph Bergh in 1895 solely on tooth characters of one specimen from the Philippines. Samples from other widely scattered Pacific localities revealed only typical C. ebraeus gene sequences. Both forms occurred in Seychelles (western Indian Ocean), where their radular teeth and diets were consistent with the data from Okinawa, but DNA of available material was degraded. Although C. judaeus was long dismissed as an aberrant specimen and junior synonym of C. ebraeus, our results support its validity as a distinct species. These results highlight the importance of molecular and radular tooth characters relative to those of the shell. Moreover, cryptic species could well be important components of species richness in Conus specifically and marine molluscan biodiversity more generally.

Background: Members of the predatory gastropod genus Conus use a venom comprised of a cocktail of peptide neurotoxins, termed conotoxins or conopeptides, to paralyze prey and conotoxin gene family members diversify via strong positive selection. Because Conus venoms are used primarily to subdue prey, the evolution of venoms is likely affected by predator-prey interactions., Methodology/principal Findings: To identify the selective forces that drive the differentiation of venoms within species of Conus, we examined the distribution of alleles of a polymorphic O-superfamily conotoxin locus of Conus ebraeus at Okinawa, Guam and Hawaii. Previous analyses of mitochondrial cytochrome oxidase I gene sequences suggest that populations of C. ebraeus, a worm-eating Conus, are not structured genetically in the western and central Pacific. Nonetheless, because the sample size from Guam was relatively low, we obtained additional data from this location and reexamined patterns of genetic variation at the mitochondrial gene at Okinawa, Guam and Hawaii. We also utilized a DNA-based approach to identify prey items of individuals of C. ebraeus from Guam and compared this information to published data on diets at Okinawa and Hawaii. Our results show that conotoxin allelic frequencies differ significantly among all three locations, with strongest differentiation at Hawaii. We also confirm previous inferences that C. ebraeus exhibits no genetic differentiation between Okinawa, Guam and Hawaii at the mitochondrial locus. Finally, DNA-based analyses show that eunicid polychaetes comprise the majority of the prey items of C. ebraeus at Guam; while this results compares well with observed diet of this species at Okinawa, C. ebraeus preys predominantly on nereid polychaetes at Hawaii., Conclusions/significance: These results imply that strong selection pressures affect conotoxin allelic frequencies. Based on the dietary information, the selection may derive from geographic variation in dietary specialization and local coevolutionary arms races between Conus and their prey.

AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence diversification mechanisms in Conus species.

Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus ("cone snails"). Cone snail biodiversity was exploited to identify three new mu-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous mu-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na(v)1.4. Our preliminary results indicate that BuIIIB and BuIIIC are potent inhibitors of Na(v)1.4 (average block approximately 96%, at a 1muM concentration of peptide), displaying a very slow off-rate not seen in previously characterized mu-conotoxins that block Na(v)1.4. In addition, the three new C. bullatus mu-conopeptides help to define a new branch of the M-superfamily of conotoxins, namely M-5. The exogene strategy used to discover these Na channel-inhibiting peptides was based on both understanding the phylogeny of Conus, as well as the molecular genetics of venom mu-conotoxin peptides previously shown to generally target voltage-gated Na channels. The discovery of BuIIIA, BuIIIB and BuIIIC Na channel blockers expands the diversity of ligands useful in determining the structure-activity relationship of voltage-gated sodium channels.

The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. alpha3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of alpha3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences of alpha3/5 conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the alpha1-delta subunit interface instead of the alpha1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.

In the four decades since toxinologists in Australia and elsewhere started to investigate the active constituents of venomous cone snails, a wealth of information has emerged on the various classes of peptides and proteins that make their venoms such potent bioactive cocktails. This article provides an overview of the current state of knowledge of these venom constituents, several of which are of interest as potential human therapeutics as a consequence of their high potency and exquisite target specificity. With the promise of as many as 50,000 venom components across the entire Conus genus, many more interesting peptides can be anticipated.

A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.

Most of the more than 500 species of predatory marine snails in the genus Conus are tropical or semitropical, and nearly all are thought to be highly selective regarding type of prey. Conus californicus Hinds, 1844, is unusual in that it is endemic to the North American Pacific coast and preys on a large variety of benthic organisms, primarily worms and other molluscs, and also scavenges. We studied the feeding behavior of C. californicus in captivity and found that it regularly killed and consumed live prickleback fishes (Cebidichthys violaceus and Xiphister spp.). Predation involved two behavioral methods similar to those employed by strictly piscivorous relatives. One method utilized stings delivered by radular teeth; the other involved engulfing the prey without stinging. Both methods were commonly used in combination, and individual snails sometimes employed multiple stings to subdue a fish. During the course of the study, snails became aroused by the presence of live fish more quickly, as evidenced by more rapid initiation of hunting behavior. Despite this apparent adaptation, details of prey-capture techniques and effectiveness of stings remained similar over the same period.