Venom duct origins of prey capture and defensive conotoxins in piscivorous Conus striatus.

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.

Human injuries and fatalities due to venomous marine snails of the family Conidae.

OBJECTIVE: This paper provides the first compilation in more than 30 years of human injuries and fatalities from envenomation by marine gastropod molluscs of the predominantly tropical family Conidae. It seeks to apply recent advances in knowledge of the physiological effects of conopeptides and molecular genetics to improve our understanding of the human responses to stings by species that normally use their venom peptides to paralyze and overcome prey such as polychaete worms, other gastropod molluscs, and fishes. RESULTS: A database has been constructed for the 139 cases accepted as reliably reporting each human injury. It includes data on the species responsible, the time and place where the stinging occurred and the sting site on the victim's body, the time-course of clinical effects, treatment carried out, if any, and outcome. Members of the hyperdiverse genus Conus caused all the injuries, except for 2 cases involving species from the recently separated genus Conasprella. Death occurred in 36 cases, 57 cases presented with serious symptoms but recovered completely, and in 44 cases victims were only minimally affected. A few cases are listed as tentative because the information in the reports was limited or unverifiable. Many cases have undoubtedly gone unreported and been forgotten. No cases are known for the period between the date of the first reliable report in the 17th century, and the mid-19th century. Knowledge of conopeptide molecular structure and function has recently burgeoned, permitting initial exploration of relationships between the symptoms and outcomes of human injuries and modes of action of these mainly small, very toxic neuroactive peptides. These relationships are reviewed here, especially in regard to the severe and fatal cases, with the aim of making recent knowledge accessible to clinicians and others involved in treating the effects of human stings, which continue to be reported. CONCLUSIONS: Conus geographus, a specialized predator of fishes, which it paralyzes with its venom and swallows whole, is the most dangerous species to humans. It accounts for about half of the known human envenomations and almost all the fatalities. Children succumb more often to C. geographus stings than adults and stings by larger snails are lethal more often than stings from smaller snails, regardless of the victim's age. Other piscivorous Conus species have stung humans, but with nonlethal results. A few species that normally prey on other gastropods have also seriously injured humans, but most of the fatalities reported have not been confirmed. Most species of Conidae prey only on marine worms; 18 of these species are known to have stung humans, with generally mild effects. Research on the treatment of Conus stings has lagged behind that on the application of conopeptides in pharmacological research and in the development of new pharmaceuticals. However, improved communication and availability of medical aid in remote tropical areas has likely contributed to reducing the mortality rate during the last half century.

Cone shell envenomation: epidemiology, pharmacology and medical care.

The marine environment presents much danger, specifically in regards to the numerous venomous inhabitants within tropical and subtropical waters. The toxins from one such group of venomous marine snails, commonly referred to as 'cone snails', have been well documented in causing human fatalities. Yet information regarding medical treatment for cone snail envenomation is limited and poorly accessible. To correct this, medical and scientific expertise and literary review on Conus provide a basic and comprehensive directive focused on the medical treatment and post-mortem investigative analysis of cone snail envenomation. We emphasize what we expect to be the most lethal feeding group of Conus and provide a brief background to the epidemiology of their stings. We describe the venom apparatus of Conus and its utility of rapid venom delivery. We have compiled the documented incidences of Conus envenomation to offer thorough reference of known signs and symptoms - this too drawing on personal experiences in the field. We have also made available a brief background to the biochemistry and pharmacology of Conus venoms to highlight their complex nature.

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks.

Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus.

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.

Glowing seashells: diversity of fossilized coloration patterns on coral reef-associated cone snail (Gastropoda: Conidae) shells from the Neogene of the Dominican Republic.

The biology of modern Conidae (cone snails)--which includes the hyperdiverse genus Conus--has been intensively studied, but the fossil record of the clade remains poorly understood, particularly within an evolutionary framework. Here, ultraviolet light is used to reveal and characterize the original shell coloration patterns of 28 species of cone snails from three Neogene coral reef-associated deposits from the Cibao Valley, northern Dominican Republic. These fossils come from the upper Miocene Cercado Fm. and lower Pliocene Gurabo Fm., and range in age from about 6.6-4.8 Ma. Comparison of the revealed coloration patterns with those of extant species allow the taxa to be assigned to three genera of cone snails (Profundiconus, Conasprella, and Conus) and at least nine subgenera. Thirteen members of these phylogenetically diverse reef faunas are described as new species. These include: Profundiconus? hennigi, Conasprella (Ximeniconus) ageri, Conus anningae, Conus lyelli, Conus (Atlanticonus?) franklinae, Conus (Stephanoconus) gouldi, Conus (Stephanoconus) bellacoensis, Conus (Ductoconus) cashi, Conus (Dauciconus) garrisoni, Conus (Dauciconus?) zambaensis, Conus (Spuriconus?) kaesleri, Conus (Spuriconus?) lombardii, and Conus (Lautoconus?) carlottae. Each of the three reef deposits contain a minimum of 14-16 cone snail species, levels of diversity that are similar to modern Indo-Pacific reef systems. Finally, most of the 28 species can be assigned to modern clades and thus have important implications for understanding the biogeographic and temporal histories of these clades in tropical America.

Evolution at a different pace: distinctive phylogenetic patterns of cone snails from two ancient oceanic archipelagos.

Ancient oceanic archipelagos of similar geological age are expected to accrue comparable numbers of endemic lineages with identical life history strategies, especially if the islands exhibit analogous habitats. We tested this hypothesis using marine snails of the genus Conus from the Atlantic archipelagos of Cape Verde and Canary Islands. Together with Azores and Madeira, these archipelagos comprise the Macaronesia biogeographic region and differ remarkably in the diversity of this group. More than 50 endemic Conus species have been described from Cape Verde, whereas prior to this study, only two nonendemic species, including a putative species complex, were thought to occur in the Canary Islands. We combined molecular phylogenetic data and geometric morphometrics with bathymetric and paleoclimatic reconstructions to understand the contrasting diversification patterns found in these regions. Our results suggest that species diversity is even lower than previously thought in the Canary Islands, with the putative species complex corresponding to a single species, Conus guanche. One explanation for the enormous disparity in Conus diversity is that the amount of available habitat may differ, or may have differed in the past due to eustatic (global) sea level changes. Historical bathymetric data, however, indicated that sea level fluctuations since the Miocene have had a similar impact on the available habitat area in both Cape Verde and Canary archipelagos and therefore do not explain this disparity. We suggest that recurrent gene flow between the Canary Islands and West Africa, habitat losses due to intense volcanic activity in combination with unsuccessful colonization of new Conus species from more diverse regions, were all determinant in shaping diversity patterns within the Canarian archipelago. Worldwide Conus species diversity follows the well-established pattern of latitudinal increase of species richness from the poles towards the tropics. However, the eastern Atlantic revealed a striking pattern with two main peaks of Conus species richness in the subtropical area and decreasing diversities toward the tropical western African coast. A Random Forests model using 12 oceanographic variables suggested that sea surface temperature is the main determinant of Conus diversity either at continental scales (eastern Atlantic coast) or in a broader context (worldwide). Other factors such as availability of suitable habitat and reduced salinity due to the influx of large rivers in the tropical area also play an important role in shaping Conus diversity patterns in the western coast of Africa.

Geometric morphometric character suites as phylogenetic data: extracting phylogenetic signal from gastropod shells.

Despite being the objects of numerous macroevolutionary studies, many of the best represented constituents of the fossil record-including diverse examples such as foraminifera, brachiopods, and mollusks-have mineralized skeletons with limited discrete characteristics, making morphological phylogenies difficult to construct. In contrast to their paucity of phylogenetic characters, the mineralized structures (tests and shells) of these fossil groups frequently have distinctive shapes that have long proved useful for their classification. The recent introduction of methodologies for including continuous data directly in a phylogenetic analysis has increased the number of available characters, making it possible to produce phylogenies based, in whole or part, on continuous character data collected from such taxa. Geometric morphometric methods provide tools for accurately characterizing shape variation and can produce quantitative data that can therefore now be included in a phylogenetic matrix in a nonarbitrary manner. Here, the marine gastropod genus Conus is used to evaluate the ability of continuous characters-generated from a geometric morphometric analysis of shell shape-to contribute to a total evidence phylogenetic hypothesis constructed using molecular and morphological data. Furthermore, the ability of continuous characters derived from geometric morphometric analyses to place fossil taxa with limited discrete characters into a phylogeny with their extant relatives was tested by simulating the inclusion of fossil taxa. This was done by removing the molecular partition of individual extant species to produce a "cladistic pseudofossil" with only the geometric morphometric derived characters coded. The phylogenetic position of each cladistic pseudofossil taxon was then compared with its placement in the total evidence tree and a symmetric resampling tree to evaluate the degree to which morphometric characters alone can correctly place simulated fossil species. In 33-45% of the test cases (depending upon the approach used for measuring success), it was possible to place the pseudofossil taxon into the correct regions of the phylogeny using only the morphometric characters. This suggests that the incorporation of extinct Conus taxa into phylogenetic hypotheses will be possible, permitting a wide range of macroevolutionary questions to be addressed within this genus. This methodology also has potential to contribute to phylogenetic reconstructions for other major components of the fossil record that lack numerous discrete characters.

Developmental modularity and phenotypic novelty within a biphasic life cycle: morphogenesis of a cone snail venom gland.

The venom gland of predatory cone snails (Conus spp.), which secretes neurotoxic peptides that rapidly immobilize prey, is a proposed key innovation for facilitating the extraordinary feeding behaviour of these gastropod molluscs. Nevertheless, the unusual morphology of this gland has generated controversy about its evolutionary origin and possible homologues in other gastropods. I cultured feeding larvae of Conus lividus and cut serial histological sections through the developing foregut during larval and metamorphic stages to examine the development of the venom gland. Results support the hypothesis of homology between the venom gland and the mid-oesophageal gland of other gastropods. They also suggest that the mid-region of the gastropod foregut, like the anterior region, is divisible into dorsal and ventral developmental modules that have different morphological, functional and ontogenetic fates. In larvae of C. lividus, the ventral module of the middle foregut transformed into the anatomically novel venom gland of the post-metamorphic stage by rapidly pinching-off from the main dorsal channel of the mid-oesophagus, an epithelial remodelling process that may be similar to other cases where epithelial tubes and vesicles arise from a pre-existing epithelial sheet. The developmental remodelling mechanism could have facilitated an abrupt evolutionary transition to the derived morphology of this important gastropod feeding innovation.

Metamorphic remodeling of a planktotrophic larva to produce the predatory feeding system of a cone snail (Mollusca, Neogastropoda).

I used histological sections and 3D reconstructions to document development through metamorphosis of the foregut and proboscis in the conoidean neogastropod Conus lividus. A goal was to determine how highly derived features of the post-metamorphic feeding system of this gastropod predator develop without interfering with larval structures for microherbivory. A second goal was to compare foregut development in this conoidean with previous observations on foregut development in the buccinoidean neogastropod Nassarius mendicus. These two neogastropods both have a feeding larval stage, but they show major differences in post-metamorphic foregut morphology. Basic events in development of the proboscis and proboscis sheath in C. lividus and N. mendicus were similar. However, the elongate buccal tube of C. lividus forms during metamorphosis as a composite of apical epidermal tissue that grows inward and ventral foregut tissue that extends outward. The larval mouth is not carried through metamorphosis. Comparative observations on foregut development in caenogastropods, which now include data on C. lividus, suggest that the foregut incorporates dorsal and ventral modules having different ontogenetic and functional fates. This developmental modularity may have facilitated evolutionary diversification of the post-metamorphic foregut. Foregut diversification in predatory gastropods may have been further fast-tracked by developmental uncoupling of larval and post-metamorphic mouths.

Embryonic toxin expression in the cone snail Conus victoriae: primed to kill or divergent function?

Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel α- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic α-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.

Intraspecies variability and conopeptide profiling of the injected venom of Conus ermineus.

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.

Proteomic analysis provides insights on venom processing in Conus textile.

Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the Conus textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complementary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails.

Cryptic species differentiated in Conus ebraeus, a widespread tropical marine gastropod.

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.

Hidden diversity in a hyperdiverse gastropod genus: discovery of previously unidentified members of a Conus species complex.

Molecular sequence data are a powerful tool for delimiting species, particularly in cases where morphological differences are obscure. Distinguishing species in the Conus sponsalis complex of tropical marine gastropods has long been difficult, because descriptions and identification has relied exclusively on shell characters, primarily color patterns, and these often appear to intergrade among putative species. Here we use molecular sequence data from two mitochondrial gene regions (16S rRNA and cytochrome oxidase subunit I) and one nuclear locus (a four-loop conotoxin gene) to characterize the genetic discontinuity of the nominal species of this group currently accepted as valid: the Indo-West Pacific C. sponsalis, C. nanus, C. ceylanensis, C. musicus and C. parvatus, and the eastern Pacific C. nux. In these analyses C. nanus and C. sponsalis resolve quite well and appear to represent distinct evolutionary units that are mostly congruent with morphology-based distinctions. We also identified several cryptic entities whose genetic uniqueness suggests species-level distinctions. Two of these fit the original description of C. sponsalis; three forms appear to represent C. nanus but differ in adult shell size or possess a unique shell color pattern.

Replaying the tape: recurring biogeographical patterns in Cape Verde Conus after 12 million years.

Isolated oceanic islands are excellent natural laboratories to test the relative role of historical contingency and determinism in evolutionary diversification. Endemics of the marine venomous snail Conus in the Cape Verde archipelago were originated from at least two independent colonizations of 'small' and 'large' shelled species separated by 12 million years. In this study, we have reconstructed phylogenetic relationships within large-shelled Conus (C. ateralbus, C. pseudonivifer, C. trochulus, and C. venulatus) based on mitochondrial cox1 and nad4 haplotype sequences. The reconstructed molecular phylogeny revealed three well-supported and relatively divergent clades (A, B, and C) that do not correspond to current species classification based on shell colour and banding patterns. Clade A grouped specimens assigned either to C. pseudonivifer or C. trochulus, clade B is composed of specimens assigned to C. venulatus, and clade C comprises specimens assigned either to C. venulatus or C. ateralbus. Geometric morphometric analyses found significant differences between the radular teeth shape of C. pseudonivifer/C. trochulus and C. venulatus/C. ateralbus. In clades A and B, northwestern Boavista and Maio specimens cluster together to the exclusion of eastern Boavista samples. In Sal, populations form a monophyletic island assemblage (clade C). The large-shelled Conus have remarkably replicated biogeographical patterns of diversification of small-shelled Conus. Similar selective forces (i.e. nonplanktonic lecithotrophy with limited larval dispersal and allopatric diversification) together with repeated instances of low sea level stands during glacial maxima that allowed connection between islands, have overcome the effect of historical contingency, and explain the observed recurring biogeographical patterns.

Conotoxins down under.

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.

Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins.

Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species. The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.