Characterizing Venom Gene Expression, Function and Species Diversity in Predatory Marine Snails of the Terebridae

The Terebridae is a group of predatory marine snails that use their venom to feed on marine annelids. Similar to other venomous organisms, the Terebridae have evolved over millions of years, diverging from their closest relative in the Paleocene era. This thesis investigates what is driving terebrid evolution and species diversification by applying a multidimensional approach

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins

Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa

Venom Diversity and Evolution in the Most Divergent Cone Snail Genus Profundiconus

Profundiconus is the most divergent cone snail genus and its unique phylogenetic position, sister to the rest of the family Conidae, makes it a key taxon for examining venom evolution and diversity. Venom gland and foot transcriptomes of Profundiconus cf. vaubani and Profundiconus neocaledonicus were de novo assembled, annotated, and analyzed for differential expression. One hundred and thirty-seven venom components were identified from P. cf. vaubani and 82 from P. neocaledonicus, with only four shared by both species. The majority of the transcript diversity was composed of putative peptides, including conotoxins, profunditoxins, turripeptides, insulin, and prohormone-4. However, there were also a significant percentage of other putative venom components such as chymotrypsin and L-rhamnose-binding lectin. The large majority of conotoxins appeared to be from new gene superfamilies, three of which are highly different from previously reported venom peptide toxins. Their low conotoxin diversity and the type of insulin found suggested that these species, for which no ecological information are available, have a worm or molluscan diet associated with a narrow dietary breadth. Our results indicate that Profundiconus venom is highly distinct from that of other cone snails, and therefore important for examining venom evolution in the Conidae family

Using Drosophila behavioral assays to characterize terebrid venompeptide bioactivity

The number of newly discovered peptides from the transcriptomes and proteomes of animal venom arsenals is rapidly increasing, resulting in an abundance of uncharacterized peptides. There is a pressing need for a systematic, cost effective, and scalable approach to identify physiological effects of venom peptides. To address this discovery-to-function gap, we developed a sequence driven:activity-based hybrid approach for screening venom peptides that is amenable to large-venom peptide libraries with minimal amounts of peptide. Using this approach, we characterized the physiological and behavioral phenotypes of two peptides from the venom of predatory terebrid marine snails, teretoxins Tv1 from Terebra variegata and Tsu1.1 from Terebra subulata. Our results indicate that Tv1 and Tsu1.1 have distinct bioactivity. Tv1 (100 µM) had an antinociceptive effect in adult Drosophila using a thermal nociception assay to measure heat avoidance. Alternatively, Tsu1.1 (100 µM) increased food intake. These findings describe the first functional bioactivity of terebrid venom peptides in relation to pain and diet and indicate that Tv1 and Tsu1.1 may, respectively, act as antinociceptive and orexigenic agents. Tv1 and Tsu1.1 are distinct from previously identified venom peptides, expanding the toolkit of peptides that can potentially be used to investigate the physiological mechanisms of pain and diet

Molecular Diversity and Gene Evolution of the Venom Arsenal of Terebridae Predatory Marine Snails

Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails,which also includes cone snails and turrids. Characterization of cone snail venompeptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homologybased searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, suchas actinoporin, commonly foundinvenoms. This research provides novel insights intovenomevolutionandrecruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution

Diet Diversity in Carnivorous Terebrid Snails Is Tied to the Presence and Absence of a Venom Gland

Predator-prey interactions are thought to play a driving role in animal evolution, especially for groups that have developed venom as their predatory strategy. However, how the diet of venomous animals influences the composition of venom arsenals remains uncertain. Two prevailing hypotheses to explain the relationship between diet and venom composition focus on prey preference and the types of compounds in venom, and a positive correlation between dietary breadth and the number of compounds in venom. Here, we examined venom complexity, phylogenetic relationship, collection depth, and biogeography of the Terebridae (auger snails) to determine if repeated innovations in terebrid foregut anatomy and venom composition correspond to diet variation. We performed the first molecular study of the diet of terebrid marine snails by metabarcoding the gut content of 71 terebrid specimens from 17 species. Our results suggest that the presence or absence of a venom gland is strongly correlated with dietary breadth. Specifically, terebrid species without a venom gland displayed greater diversity in their diet. Additionally, we propose a revision of the definition of venom complexity in conoidean snails to more accurately capture the breadth of ecological influences. These findings suggest that prey diet is an important factor in terebrid venom evolution and diversification and further investigations of other understudied organisms, like terebrids, are needed to develop robust hypotheses in this area

Macroevolutionary Analyses Suggest That Environmental Factors, Not Venom Apparatus, Play Key Role in Terebridae Marine Snail Diversification

How species diversification occurs remains an unanswered question in predatory marine invertebrates, such as sea snails of the family Terebridae. However, the anatomical disparity found throughput the Terebridae provides a unique perspective for investigating diversification patterns in venomous predators. In this study, a new dated molecular phylogeny of the Terebridae is used as a framework for investigating diversification of the family through time, and for testing the putative role of intrinsic and extrinsic traits, such as shell size, larval ecology, bathymetric distribution, and anatomical features of the venom apparatus, as drivers of terebrid species diversification. Macroevolutionary analysis revealed that when diversification rates do not vary across Terebridae clades, the whole family has been increasing its global diversification rate since 25 Ma. We recovered evidence for a concurrent increase in diversification of depth ranges, while shell size appeared to have undergone a fast divergence early in terebrid evolutionary history. Our data also confirm that planktotrophy is the ancestral larval ecology in terebrids, and evolutionary modeling highlighted that shell size is linked to larval ecology of the Terebridae, with species with long-living pelagic larvae tending to be larger and have a broader size range than lecithotrophic species. Although we recovered patterns of size and depth trait diversification through time and across clades, the presence or absence of a venom gland (VG) did not appear to have impacted Terebridae diversification. Terebrids have lost their venom apparatus several times and we confirm that the loss of a VG happened in phylogenetically clustered terminal taxa and that reversal is extremely unlikely. Our findings suggest that environmental factors, and not venom, have had more influence on terebrid evolution

Phylogenetic classification of the family Terebridae (Neogastropoda: Conoidea)

The conoidean family Terebridae is an intriguing lineage of marine gastropods, which are of considerable interest due to their varied anatomy and complex venoms. Terebrids are abundant, easily recognizable and widely distributed in tropical and subtropical waters, but our findings have demonstrated that their systematics requires revision. Here we elaborate the classification of Terebridae based on a recently published molecular phylogeny of 154 species, plus characters of the shell and anterior alimentary system. The 407 living species of the family, including seven species described herein, are assigned to three subfamilies: Pellifroniinae new subfamily, Pervicaciinae and Terebrinae. The Pellifroniinae comprises five deep -water species in two genera, Pellifronia and Bathyterebra n. gen. Pellifroniinae possess a radula of duplex marginal teeth, well-developed proboscis and venom gland, and a very small rhynchodeal introvert. The Pervicaciinae includes c. 50 species in the predominantly Indo-Pacific genera Duplicaria and Partecosta. Pervicaciinae possess salivary glands, a radula of solid recurved marginal teeth and a weakly developed rhynchodeal introvert, but lack proboscis and venom gland. The remaining Terebridae species are classified into 15 genera in the subfamily Terebrinae (including four genera described herein); nine genera are defined on the basis of phylogenetic data and six solely on shell morphology. The Indo-Pacific genera Profitnditerebra n. gen., Maculauger n. gen. and Myurellobsis n. gen. each include about a dozen species. The first is restricted to the deep waters of the Indo-West Pacific, while the latter two range widely in both geographic and bathymetric distribution. Neoterebra n. gen. encompasses about 65 species from a range of localities in the eastern Pacific, Caribbean, and Atlantic, and from varying depths. To characterize the highly diversified genera Terebra, Punctoterebra, Myurella and Dublicaria, each of which comprise several morphological clusters, we propose the use of DNA-based diagnoses. These diagnoses are combined with more informative descriptions to define most of the supraspecific taxa of Terebridae, to provide a comprehensive revision of the group

From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins

Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa

Venom Diversity and Evolution in the Most Divergent Cone Snail Genus Profundiconus

Profundiconus is the most divergent cone snail genus and its unique phylogenetic position, sister to the rest of the family Conidae, makes it a key taxon for examining venom evolution and diversity. Venom gland and foot transcriptomes of Profundiconus cf. vaubani and Profundiconus neocaledonicus were de novo assembled, annotated, and analyzed for differential expression. One hundred and thirty-seven venom components were identified from P. cf. vaubani and 82 from P. neocaledonicus, with only four shared by both species. The majority of the transcript diversity was composed of putative peptides, including conotoxins, profunditoxins, turripeptides, insulin, and prohormone-4. However, there were also a significant percentage of other putative venom components such as chymotrypsin and L-rhamnose-binding lectin. The large majority of conotoxins appeared to be from new gene superfamilies, three of which are highly different from previously reported venom peptide toxins. Their low conotoxin diversity and the type of insulin found suggested that these species, for which no ecological information are available, have a worm or molluscan diet associated with a narrow dietary breadth. Our results indicate that Profundiconus venom is highly distinct from that of other cone snails, and therefore important for examining venom evolution in the Conidae family