Predatory Marine Gastropods Research Articles

The Characterization of the Mitochondrial Genome of Fulgoraria rupestris and Phylogenetic Considerations within the Neogastropoda.

Fulgoraria rupestris is a predatory marine gastropod belonging to Neogastropoda and possessing considerable taxonomic significance. However, research on this species remains limited. We acquired the complete mitochondrial genome of F. rupestris through second-generation sequencing and conducted an analysis of its genome structural features. The mitochondrial genome of F. rupestris spans a total length of 16,223 bp and encompasses 37 genes (13 protein-coding genes (PCGs), 22 transfer RNAs, and 2 ribosomal RNAs). Notably, most tRNAs exhibit the typical cloverleaf structure, but there is an absence of the Dihydrouridine (DHU) arm in the trnS1 and trnS2 genes. The A + T content is 68.67%, indicating a pronounced AT bias. Additionally, we conducted a selection pressure analysis on the mitochondrial genomes of four species within Volutidae, revealing that all PCGs are subjected to purifying selection. In comparison to other species within Neogastropoda, F. rupestris shares an identical gene arrangement. Additionally, based on mitochondrial genome sequences of the 13 PCGs from 50 species within Neogastropoda, we constructed a phylogenetic tree. The phylogenetic tree indicates F. rupestris forms a clade with species within the family Volutidae (Cymbium olla, Neptuneopsis gilchristi, and Melo melo). This study serves as a valuable reference for future research on F. rupestris, offering insights for the upcoming phylogenetic and taxonomic classification within Neogastropoda. Furthermore, the findings provide valuable information for the development of genetic resources in this context.

Temperature-dependent responses and trophic interaction strengths of a predatory marine gastropod and rock oyster under ocean warming

Predator-prey interactions are important in shaping ecosystem structure. Consequently, impacts of accelerating global warming on predators will have notable implications. Effects are likely to be particularly marked for tropical organisms which are anticipated to be sensitive to further thermal stress. Here, we investigated effects of future ocean warming on the predatory dogwhelk Reishia clavigera and its predation of Saccostrea cucullata. Mortality of the predators rapidly increased under the extreme elevated temperature, while those exposed to moderate elevated temperature displayed similar mortality as the ambient. Predators that survived moderate temperature increases altered their oxygen consumption patterns, increased average feeding rates, and functional responses, although condition index and energy reserves were unchanged. Overall, we show extreme ocean warming scenarios can remove predators and their consumption of prey from an ecosystem, whereas moderate warming can intensify predator-prey interactions. Such temperature-dependent alterations to predator-prey interactions would lead to fundamental changes of ecosystem structure as the ocean warms.

Ocean Warming and Heat Stress Impact Molecules of Keystone Significance in a Predatory Marine Gastropod

Water temperature is a major abiotic driver, controlling the rates and nature of biochemical reactions and subsequently affecting the physiology of marine organisms. However, relatively little is known about the implications of heat stress or predicted ocean climate change on marine secondary metabolites. The predatory gastropod Dicathais orbita is a useful model organism for climate change and natural product studies. Here we determine the upper thermal limit (CTMax) of D. orbita and investigate the effects of thermal stress on the bioactive compounds stored in their biosynthetic organ, the hypobranchial gland. Two CTMax experiments were undertaken, along with a static heat stress experiment where whelks were exposed to an elevated temperature of 30°C for one week, compared to a 20°C seawater control. An additional 35-day ocean climate change experiment used combinations of temperature (ambient: 23°C and future: 25°C) and pCO2 (ambient: ~380 ppm and future: ~765 ppm). The impacts on secondary metabolites in all experiments were assessed using liquid chromatography-mass spectrometry. The mean CTMax of the whelks, from the northern limit of their distribution, was found to be 35.2°C using a rapid temperature increase rate of 1°C/1 h, but was only 30.6°C when a gradual heating rate of 1°C/12 h was used. The overall composition of the secondary metabolites was significantly affected by heat stress in all four experiments, but not by elevated pCO2 in the ocean climate change experiment. The proportion of the choline ester murexine was significantly reduced in heat-stressed snails compared to the controls. Tyrindoxyl sulphate was significantly reduced under prolonged exposure to future temperature, whereas the relative abundance of the oxidation product, 6-bromoisatin significantly increased with elevated temperature exposure. Despite the fact that intertidal gastropods like D. orbita might be able to buffer the impact of external temperatures within the predicted future range, this study provides evidence that ocean warming could have significant implications for secondary metabolite production and/or storage in marine invertebrates. Impacts on bioactive molecules with multifunctional ecological roles could have implications for predator populations with possible flow on effects in some marine communities.

Diversity of Conidae (Caenogastropoda: Conoidea) from the Kerala Coast, India

ABSTRACT The Conidae are predatory marine gastropods and they produce one of the most potent venoms in nature. They are ecologically vital for regulating trophic positions and act as primary carnivores in oceans. Amateur shell collectors target some species and ecosystems are intensively exploited by collectors of ornamental shells and commercial traders. Moreover, increasing coastal development, habitat modification and fragmentation, and overexploitation of resources threaten cone snail diversity. This study documents 46 species of Conidae from the Kerala coast, India. Fifteen species are newly recorded from the coast, and one species is reported for the first time from the Indian coast. We also revalidate Conus ceylanensis and Conus bizona.

Densities of Large Marine Gastropods in Seagrass, Oyster Reef, and Sandy Habitats in Tampa Bay and along the Gulf Coast of Florida

Marine gastropods in Florida are harvested by the marine-life trade, tourists (for shells), and traditional commercial fisheries, but stocks are not formally assessed for most species. The main goal of this project was to estimate biological variables that may be used to evaluate the Species of Greatest Conservation Need status of the banded tulip Cinctura lilium and six other large marine gastropod species that are commonly encountered in the targeted habitats. The principal target, C. lilium, was listed in the 2012 State Wildlife Action Plan. The densities and size structures of C. lilium and six co-occurring species of large marine gastropods in Tampa Bay were examined to evaluate population dynamics, seasonal variation, and habitat preferences of these species. Long-term trends in the densities of large, predatory marine gastropods in 10 regions from 2009 to 2017 were included. In Tampa Bay, strong habitat preferences were observed for all species studied: C. lilium and Melongena corona were most likely to be observed on oyster reefs, and Fasciolaria tulipa was found only in seagrass, whereas Strombus alatus, Triplofusus gigantea, Sinistrofulgur sinistrum, and Fulguropsis spirata were more common on soft sediments. Seasonal variations in densities were weak; seasonal differences in size structure were more pronounced. The smallest individuals of C. lilium appeared in spring and summer; F. spirata, S. alatus, and S. sinistrum reached a detectable size in fall. The smallest detectable M. corona appeared in winter. Regionally, there were more gastropods in southwestern bay grass beds (Tampa Bay, Sarasota Bay, and Pine Island Sound) than in coastal seagrass beds (Big Bend, Citrus, Hernando, Pasco, and coastal Pinellas) or northern bays (St. Joseph Bay and St. Andrew Bay), and each region had characteristic species. As a group, tulip snails exhibited modest local declines in densities at two sites, whereas the whelks and T. gigantea varied annually but did not exhibit consistent trends during 2009 to 2017. The present study establishes a baseline of abundance for seven species of common marine gastropods and recommends expanded monitoring in multiple habitats to improve future analyses of abundance trends.

Acoustic tracking of a large predatory marine gastropod, Charonia tritonis, on the Great Barrier Reef

Crown-of-thorns starfish Acanthaster planci (COTS) outbreaks are a major cause of coral cover loss on the Great Barrier Reef (GBR), with manual culling having only localised success. The endangered giant triton snail Charonia tritonis is a natural predator of COTS, although aquarium and field observations indicate the intensity of direct predation may be inadequate to significantly mediate outbreaks. However, their mere presence can elicit a chemically induced sensory behavioural response which may suppress COTS populations when in non-outbreak status. While there is increasing knowledge of the sensory biology of both species, little is known regarding giant triton snail numbers on the GBR or about how they move and occupy space, making it difficult to determine their true zone of influence and thus their capacity to disrupt COTS behaviour. We used passive acoustic telemetry to establish short-term activity space and movement patterns of giant triton snails on the GBR. Individuals were tracked for up to 41 d, were observed to travel 234.24 m d-1, with a mean total cumulative distance travelled at night (1923.19 m) nearly double that observed during the day (1014.84 m). These distances encompass those reported for COTS (10.3 m d-1) and align with COTS nocturnal behaviour. Space utilisation distributions (UDs) revealed a mean (±SD) home range of 1179.40 ± 659.40 m2 (95% UD) and a core area of 195.68 ± 141.31 m2 (50% UD). Revealing the short-term movement patterns of this natural COTS predator within a reef environment advances knowledge of its spatial ecology and will provide information for its future conservation and for COTS management efforts.

Mitogenomic phylogeny of the Naticidae (Gastropoda: Littorinimorpha) reveals monophyly of the Polinicinae

AbstractThe Naticidae is a species‐rich family of predatory marine gastropods with substantial interspecific morphological diversity. The classification of the Naticidae has been traditionally based on morphology data, but the phylogenetic relationships within the family are debated due to conflicting molecular results, especially regarding the monophyly of subfamilies Polinicinae and Naticinae. To further resolve the phylogenetic controversies within the Naticidae, we undertake a phylogenetic approach using 14 newly sequenced complete or nearly complete (only lacking a control region) mitochondrial genomes. Both the maximum likelihood and Bayesian inference analyses supported monophyly of the Polinicinae, but paraphyly of the Naticinae due to the placement of the enigmatic genus Notocochlis. The ancestral character reconstruction suggests that the operculum, a character that currently defines the two subfamilies, evolved from an ancestor with a calcareous operculum in the evolutionary history of naticids. In addition, the chronogram estimates that naticids was originated in late Triassic (about 227 million years ago), consistent with previous hypotheses. Our study highlights the importance of using complete mitochondrial genomes while reconstructing phylogenetic relationships within the Naticidae. The evolution scenario of the naticid operculum contributes new insights into the classification of Naticidae.

Bioactive Compounds Isolated from Neglected Predatory Marine Gastropods.

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.

Application of community phylogenetic approaches to understand gene expression: differential exploration of venom gene space in predatory marine gastropods.

BackgroundPredatory marine gastropods of the genus Conus exhibit substantial variation in venom composition both within and among species. Apart from mechanisms associated with extensive turnover of gene families and rapid evolution of genes that encode venom components (‘conotoxins’), the evolution of distinct conotoxin expression patterns is an additional source of variation that may drive interspecific differences in the utilization of species’ ‘venom gene space’. To determine the evolution of expression patterns of venom genes of Conus species, we evaluated the expression of A-superfamily conotoxin genes of a set of closely related Conus species by comparing recovered transcripts of A-superfamily genes that were previously identified from the genomes of these species. We modified community phylogenetics approaches to incorporate phylogenetic history and disparity of genes and their expression profiles to determine patterns of venom gene space utilization.ResultsLess than half of the A-superfamily gene repertoire of these species is expressed, and only a few orthologous genes are coexpressed among species. Species exhibit substantially distinct expression strategies, with some expressing sets of closely related loci (‘under-dispersed’ expression of available genes) while others express sets of more disparate genes (‘over-dispersed’ expression). In addition, expressed genes show higher dN/dS values than either unexpressed or ancestral genes; this implies that expression exposes genes to selection and facilitates rapid evolution of these genes. Few recent lineage-specific gene duplicates are expressed simultaneously, suggesting that expression divergence among redundant gene copies may be established shortly after gene duplication.ConclusionsOur study demonstrates that venom gene space is explored differentially by Conus species, a process that effectively permits the independent and rapid evolution of venoms in these species.

Morphology of sensory papillae on the feeding proboscis of cone snails (Mollusca, Gastropoda)

AbstractCone snails, predatory marine gastropods, have developed a specialized prey capture method in which a long, distensible proboscis is used to identify prey and inject venom via a hydraulically propelled hollow radular tooth. Using brightfield, epifluorescence, confocal, and transmission and scanning electron microscopy, we describe the morphology of ciliated sensory structures concentrated on the tip of the proboscis. The number and morphology of these sensory papillae are linked to the type of preferred prey: cone snails feeding on worms and mollusks have short, cone or finger‐shaped papillae, whereas fish‐hunting cone snails have long tubular papillae in addition to short conical papillae. Sensory papillae are well positioned to provide information necessary to locate, identify, and dispatch prey. Proboscis tips and their sensory papillae regenerated within 10 d following experimental ablation, and snails with regenerated proboscis tips were able to locate and envenomate prey. The remarkable intrageneric variation found in the morphology of these sensory structures is probably linked to the specialized prey that cone snail species have evolved to hunt.

Trophic diversification in the evolution of predatory marine gastropods of the family Terebridae as inferred from stable isotope data

Trophic diversification in the evolution of predatory marine gastropods of the family Terebridae as inferred from stable isotope data

Molecular Evolution and Diversity of Conus Peptide Toxins, as Revealed by Gene Structure and Intron Sequence Analyses

Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.

Natural Product Research in the Australian Marine Invertebrate Dicathais orbita

The predatory marine gastropod Dicathais orbita has been the subject of a significant amount of biological and chemical research over the past five decades. Natural products research on D. orbita includes the isolation and identification of brominated indoles and choline esters as precursors of Tyrian purple, as well as the synthesis of structural analogues, bioactivity testing, biodistributional and biosynthetic studies. Here I also report on how well these compounds conform to Lipinski’s rule of five for druglikeness and their predicted receptor binding and enzyme inhibitor activity. The composition of mycosporine-like amino acids, fatty acids and sterols has also been described in the egg masses of D. orbita. The combination of bioactive compounds produced by D. orbita is of interest for further studies in chemical ecology, as well as for future nutraceutical development. Biological insights into the life history of this species, as well as ongoing research on the gene expression, microbial symbionts and biosynthetic capabilities, should facilitate sustainable production of the bioactive compounds. Knowledge of the phylogeny of D. orbita provides an excellent platform for novel research into the evolution of brominated secondary metabolites in marine molluscs. The range of polarities in the brominated indoles produced by D. orbita has also provided an effective model system used to develop a new method for biodistributional studies. The well characterized suite of chemical reactions that generate Tyrian purple, coupled with an in depth knowledge of the ecology, anatomy and genetics of D. orbita provide a good foundation for ongoing natural products research.

Evolution of the Radular Apparatus in Conoidea (Gastropoda: Neogastropoda) as Inferred from a Molecular Phylogeny

The anatomy and evolution of the radular apparatus in predatory marine gastropods of the superfamily Conoidea is reconstructed on the basis of a molecular phylogeny, based on three mitochondrial genes (COI, 12S and 16S) for 102 species. A unique feeding mechanism involving use of individual marginal radular teeth at the proboscis tip for stabbing and poisoning of prey is here assumed to appear at the earliest stages of evolution of the group. The initial major evolutionary event in Conoidea was the divergence to two main branches. One is characterized by mostly hypodermic marginal teeth and absence of an odontophore, while the other possesses a radula with primarily duplex marginal teeth, a strong subradular membrane and retains a fully functional odontophore. The radular types that have previously been considered most ancestral, “prototypic” for the group (flat marginal teeth; multicuspid lateral teeth of Drilliidae; solid recurved teeth of Pseudomelatoma and Duplicaria), were found to be derived conditions. Solid recurved teeth appeared twice, independently, in Conoidea – in Pseudomelatomidae and Terebridae. The Terebridae, the sister group of Turridae, are characterized by very high radular variability, and the transformation of the marginal radular teeth within this single clade repeats the evolution of the radular apparatus across the entire Conoidea.

Proteomic Interrogation of Venom Delivery in Marine Cone Snails: Novel Insights into the Role of the Venom Bulb

Cone snails of the genus Conus are predatory marine gastropods mainly found in the shallow waters of the tropics and warm temperate seas. To prey on other marine organisms including fish, cone snails have evolved complex venoms synthesized and delivered by a highly sophisticated venom apparatus. Upon prey discovery, the venom is perfused through a harpoon-like radula tooth and rapidly injected into the prey to cause paralysis. While the venom components of cone snails have been intensively characterized, the mechanism of venom translocation and loading prior to and during injection remains elusive. The involvement of the venom bulb, a muscular dilation of the venom gland has been suggested, however evidence is sparse. Here, we use a combination of proteomics, molecular biology, and morphological examination to elucidate the potential role of the venom bulb in venom translocation and delivery. Analysis of the venom bulb proteome clearly demonstrated a function of this organ in muscular movement and, more interestingly, in burst muscle contraction. Morphological examination revealed high structural similarities to the mantle muscle of squids, animals known for their rapid escape response. We sequenced and further characterized arginine kinase, a key protein of rapid muscular movement in invertebrates and show high concentrations of this enzyme in the bulb when compared to the venom gland and the foot muscle. Proteins characteristic for venom biosynthesis were low in abundance. On the basis of our findings, we suggest that the bulb of cone snails is a highly specialized organ of venom translocation. Delivery of venom is driven by burst contractions of the bulb rapidly forcing the venom through the radula tooth into the prey.

Geographic Variation in Venom Allelic Composition and Diets of the Widespread Predatory Marine Gastropod Conus ebraeus

BackgroundMembers 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 FindingsTo 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/SignificanceThese 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.

Ecological Release and Venom Evolution of a Predatory Marine Snail at Easter Island

BackgroundEcological release is coupled with adaptive radiation and ecological diversification yet little is known about the molecular basis of phenotypic changes associated with this phenomenon. The venomous, predatory marine gastropod Conus miliaris has undergone ecological release and exhibits increased dietary breadth at Easter Island.Methodology/Principal FindingsWe examined the extent of genetic differentiation of two genes expressed in the venom of C. miliaris among samples from Easter Island, American Samoa and Guam. The population from Easter Island exhibits unique frequencies of alleles that encode distinct peptides at both loci. Levels of divergence at these loci exceed observed levels of divergence observed at a mitochondrial gene region at Easter Island.Conclusions/SignificancePatterns of genetic variation at two genes expressed in the venom of this C. miliaris suggest that selection has operated at these genes and contributed to the divergence of venom composition at Easter Island. These results show that ecological release is associated with strong selection pressures that promote the evolution of new phenotypes.

Genetically monomorphic invasive populations of the rapa whelk, Rapana venosa

Rapana venosa is a predatory marine gastropod native to the coastal waters of China, Korea, and Japan. Since the 1940s, R. venosa has been transported around the globe and introduced populations now exist in the Black Sea, the Mediterranean Sea, the Adriatic and Aegean seas, off the coasts of France and The Netherlands, in Chesapeake Bay, Virginia, USA, and in the Rio de la Plata between Uruguay and Argentina. This study surveyed variation in two mitochondrial gene regions to investigate the invasion pathways of R. venosa, identify likely sources for introduced populations, and evaluate current hypotheses of potential transportation vectors. Sequence data were obtained for the mitochondrial cytochrome c oxidase I and NADH dehydrogenase subunit 2 gene regions of 178 individuals from eight native locations and 106 individuals from 12 introduced locations. Collections from within the native range displayed very high levels of genetic variation while collections from all introduced populations showed a complete lack of genetic diversity; a single haplotype was common to all introduced individuals. This finding is consistent with the hypothesis that R. venosa was initially introduced into the Black Sea, and this Black Sea population then served as a source for the other secondary invasions by various introduction vectors including ballast water transport. Although non-native R. venosa populations currently appear to be thriving in their new environments, the lack of genetic variability raises questions regarding the evolutionary persistence of these populations.

Differentiation of Venoms of Predatory Marine Gastropods: Divergence of Orthologous Toxin Genes of Closely Related Conus Species with Different Dietary Specializations

Venoms of Conus are remarkably diverse among species and the genes that encode conotoxins show high rates of evolution. Yet no prior studies have specifically explored how conotoxin gene evolution contributes to the differentiation of venoms of closely related Conus species. Previous investigations of four-loop conotoxin expression patterns of six closely related Conus species identified 12 sets of putative orthologous loci from these species, including eight pairs of loci that are coexpressed by two of these six species, C. abbreviatus and C. miliaris. Here I analyze the molecular evolution of orthologous conotoxin loci of these species and specifically examine the divergence of the eight orthologous counterparts of C. abbreviatus and C. miliaris. Tree and maximum likelihood-based analyses of these sequences reveal that positive selection promotes the divergence of orthologous genes among species and that the evolution of orthologues of C. abbreviatus and C. miliaris is asymmetric among species. The asymmetric evolution of conotoxin loci among species may result from lineage-specific dietary shifts or interspecific differences in the impact of selection from predator-prey interactions on conotoxin loci.

Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails

Venoms of predatory marine gastropods of the genus Conus show amazing levels of interspecific diversity and are comprised of a cocktail of peptide neurotoxins, termed conotoxins, that are encoded by large gene families. Conotoxin gene family evolution is characterized by gene duplications and high rates of nonsynonymous substitution among paralogues; yet, what controls the differentiation of venoms among species is not clear. We compared four-loop conotoxin transcripts of six closely related Conus species to examine conotoxin expression patterns among species. The species examined appear to express different numbers of four-loop conotoxin loci and similarity in expression patterns does not seem to correspond with phylogenetic affinity. Moreover, several loci appear to have been independently silenced while others appear to have been revived from previously silenced states. Some loci also appear to exhibit coordinated expression patterns. These results suggest that the evolution of conotoxin expression patterns is incredibly dynamic and the differentiation of venoms of Conus is controlled in part by the evolution of unique conotoxin expression patterns.