DeTox: a pipeline for the detection of toxins in venomous organisms.

Venomous organisms have independently evolved the ability to produce toxins 101 times during their evolutionary history, resulting in over 200 000 venomous species. Collectively, these species produce millions of toxins, making them a valuable resource for bioprospecting and understanding the evolutionary mechanisms underlying genetic diversification. RNA-seq is the preferred method for characterizing toxin repertoires, but the analysis of the resulting data remains challenging. While early approaches relied on similarity-based mapping to known toxin databases, recent studies have highlighted the importance of structural features for toxin detection. The few existing pipelines lack an integration between these complementary approaches, and tend to be difficult to run for non-experienced users. To address these issues, we developed DeTox, a comprehensive and user-friendly tool for toxin research. It combines fast execution, parallelization and customization of parameters. DeTox was tested on published transcriptomes from gastropod mollusks, cnidarians and snakes, retrieving most putative toxins from the original articles and identifying additional peptides as potential toxins to be confirmed through manual annotation and eventually proteomic analysis. By integrating a structure-based search with similarity-based approaches, DeTox allows the comprehensive characterization of toxin repertoire in poorly-known taxa. The effect of the taxonomic bias in existing databases is minimized in DeTox, as mirrored in the detection of unique and divergent toxins that would have been overlooked by similarity-based methods. DeTox streamlines toxin annotation, providing a valuable tool for efficient identification of venom components that will enhance venom research in neglected taxa.

Coupling DNA barcodes and exon-capture to resolve the phylogeny of Turridae (Gastropoda, Conoidea).

Taxon sampling in most phylogenomic studies is often based on known taxa and/or morphospecies, thus ignoring undescribed diversity and/or cryptic lineages. The family Turridae is a group of venomous snails within the hyperdiverse superfamily Conoidea that includes many undescribed and cryptic species. Therefore 'traditional' taxon sampling could constitute a strong risk of undersampling or oversampling Turridae lineages. To minimize potential biases, we establish a robust sampling strategy, from species delimitation to phylogenomics. More than 3,000 cox-1 "barcode" sequences were used to propose 201 primary species hypotheses, nearly half of them corresponding to species potentially new to science, including several cryptic species. A 110-taxa exon-capture tree, including species representatives of the diversity uncovered with the cox-1 dataset, was build using up to 4,178 loci. Our results show the polyphyly of the genus Gemmula, that is split into up to 10 separate lineages, of which half would not have been detected if the sampling strategy was based only on described species. Our results strongly suggest that the use of blind, exploratory and intensive barcode sampling is necessary to avoid sampling biases in phylogenomic studies.

Collaborative Expression: Transcriptomics of Conus virgo Suggests Contribution of Multiple Secretory Glands to Venom Production.

Venomous marine gastropods of the family Conidae are among the most diversified predators in marine realm-in large due to their complex venoms. Besides being a valuable source of bioactive neuropeptides conotoxins, cone-snails venoms are an excellent model for molecular evolution studies, addressing origin of key innovations. However, these studies are handicapped by scarce current knowledge on the tissues involved in venom production, as it is generally assumed the sole prerogative of the venom gland (VG). The role of other secretory glands that are present in all Conus species (salivary gland, SG) or only in some species (accessory salivary gland, ASG) remains poorly understood. Here, for the first time, we carry out a detailed analysis of the VG, SG, and ASG transcriptomes in the vermivorous Conus virgo. We detect multiple transcripts clusters in both the SG and ASG, whose annotations imply venom-related functions. Despite the subsets of transcripts highly-expressed in the VG, SG, and ASG being very distinct, SG expresses an L-, and ASG-Cerm08-, and MEFRR- superfamily conotoxins, all previously considered specific for VG. We corroborate our results with the analysis of published SG and VG transcriptomes from unrelated fish-hunting C. geographus, and C. striatus, possibly fish-hunting C. rolani, and worm-hunting Conus quercinus. In spite of low expression levels of conotoxins, some other specific clusters of putative venom-related peptides are present and may be highly expressed in the SG of these species. Further functional studies are necessary to determine the role that these peptides play in envenomation. In the meantime, our results show importance of routine multi-tissue sampling both for accurate interpretation of tissue-specific venom composition in cone-snails, and for better understanding origin and evolution of venom peptides genes.

Raising names from the dead: A time-calibrated phylogeny of frog shells (Bursidae, Tonnoidea, Gastropoda) using mitogenomic data.

With 59 Recent species, Bursidae, known as «frog shells¼, are a small but widely distributed group of tropical and subtropical gastropods that are most diverse in the Indo-West Pacific. The present study is aimed at reconstructing phylogenetic relationships of bursid gastropods based on extensive and representative taxon sampling. Five genetic markers (cytochrome c oxidase subunit I (cox1), 16 s and 12 s rRNA mitochondrial genes, 28 s rRNA and Histone H3 nuclear gene) were sequenced for over 30 species in every known genus but Crossata. Furthermore, we sequenced the complete mt-genome of 9 species (10 specimens) (Aspa marginata, Marsupina bufo, Korrigania quirihorai, Korrigania fijiensis, Tutufa rubeta, Bursa lamarckii, Lampasopsis rhodostoma (twice), Bufonaria perelegans and Bursa aff. tuberosissima). Our analysis recovered Bursidae as a monophyletic group, whereas the genus Bursa was found to be polyphyletic. The genera Talisman and Dulcerana are resurrected and the genera Alanbeuella gen. nov. and Korrigania gen. nov. are described. Dating analysis using 21 extinct taxa for node and simplified tip calibrations was performed, showing a diversification of the group in two phases. Diversification may be linked to tectonic events leading to biodiversity relocation from the western Tethys toward the Indo-Pacific.

Frogs and tuns and tritons - A molecular phylogeny and revised family classification of the predatory gastropod superfamily Tonnoidea (Caenogastropoda).

The Tonnoidea is a moderately diverse group of large, predatory gastropods with ∼360 valid species. Known for their ability to secrete sulfuric acid, they use it to prey on a diversity of invertebrates, primarily echinoderms. Tonnoideans currently are classified in seven accepted families: the comparatively well known, shallow water Bursidae, Cassidae, Personidae, Ranellidae, and Tonnidae, and the lesser-known, deep water Laubierinidae and Pisanianuridae. We assembled a mitochondrial and nuclear gene (COI, 16S, 12S, 28S) dataset for ∼80 species and 38 genera currently recognized as valid. Bayesian analysis of the concatenated dataset recovered a monophyletic Tonnoidea, with Ficus as its sister group. Unexpectedly, Thalassocyon, currently classified in the Ficidae, was nested within the ingroup as the sister group to Distorsionella. Among currently recognized families, Tonnidae, Cassidae, Bursidae and Personidae were supported as monophyletic but the Ranellidae and Ranellinae were not, with Cymatiinae, Ranella and Charonia supported as three unrelated clades. The Laubierinidae and Pisanianuridae together form a monophyletic group. Although not all currently accepted genera have been included in the analysis, the new phylogeny is sufficiently robust and stable to the inclusion/exclusion of nonconserved regions to establish a revised family-level classification with nine families: Bursidae, Cassidae, Charoniidae, Cymatiidae, Laubierinidae, Personidae, Ranellidae, Thalassocyonidae and Tonnidae. The results reveal that many genera as presently circumscribed are para- or polyphyletic and, in some cases support the rescue of several genus-group names from synonymy (Austrosassia, Austrotriton, Laminilabrum, Lampadopsis, Personella, Proxicharonia, Tritonoranella) or conversely, support their synonymization (Biplex with Gyrineum). Several species complexes are also revealed that merit further investigation (e.g., Personidae: Distorsio decipiens, D. reticularis; Bursidae: Bursa tuberosissima; Cassidae: Echinophoria wyvillei, Galeodea bituminata, and Semicassis bisulcata). Consequently, despite their teleplanic larvae, the apparently circumglobal distribution of some tonnoidean species is the result of excessive synonymy. The superfamily is estimated to have diverged during the early Jurassic (∼186 Ma), with most families originating during a narrow ∼20 My window in Albian-Aptian times as part of the Mesozoic Marine Revolution.

Exploring the Udoteaceae diversity (Bryopsidales, Chlorophyta) in the Caribbean region based on molecular and morphological data.

The Udoteaceae family (Bryopsidales, Chlorophyta) is known to be highly diverse morphologically in the Caribbean region, but only few studies have studied its genetic diversity. Using an integrative taxonomic approach, this study aimed at (1) exploring the Udoteaceae species diversity using a combination of five DNA-based species delimitation methods and morpho-anatomical data for confirmation; (2) estimating the discriminatory power of traditional diagnostic characters using a morphology-based clustering method and statistical analyses focused on the genus Udotea; and (3) reconstructing the phylogeny of the family based on a multilocus analysis (tufA, rbcL, 18S rDNA). Our results revealed strong congruence between species hypotheses across delimitation methods and markers. Morpho-anatomical characters proved essential to validate these hypotheses, to assign species names and to unveil new species. Morphological analyses led to relevant results for accurately discriminating Udotea morphospecies. Siphon features and cortication were key characters to define supra-specific groups and to revise the taxonomy of the genus Udotea. Phylogenetic analyses confirmed the polyphyly of Udotea, Rhipocephalus and Penicillus, which led us to propose a revised definition of Udotea sensu stricto based on both genetic and morphological data. Finally, our study emphasizes the importance of combining genetic and morphological data for the taxonomic revision of the Udoteaceae, but stresses the need of including more taxa from other geographical regions to better resolve taxonomic issues.

Quantification of complex modular architecture in plants.

Morphometrics, the assignment of quantities to biological shapes, is a powerful tool to address taxonomic, evolutionary, functional and developmental questions. We propose a novel method for shape quantification of complex modular architecture in thalloid plants, whose extremely reduced morphologies, combined with the lack of a formal framework for thallus description, have long rendered taxonomic and evolutionary studies extremely challenging. Using graph theory, thalli are described as hierarchical series of nodes and edges, allowing for accurate, homologous and repeatable measurements of widths, lengths and angles. The computer program MorphoSnake was developed to extract the skeleton and contours of a thallus and automatically acquire, at each level of organization, width, length, angle and sinuosity measurements. Through the quantification of leaf architecture in Hymenophyllum ferns (Polypodiopsida) and a fully worked example of integrative taxonomy in the taxonomically challenging thalloid liverwort genus Riccardia, we show that MorphoSnake is applicable to all ramified plants. This new possibility of acquiring large numbers of quantitative traits in plants with complex modular architectures opens new perspectives of applications, from the development of rapid species identification tools to evolutionary analyses of adaptive plasticity.

Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis.

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

The contrasted evolutionary fates of deep-sea chemosynthetic mussels (Bivalvia, Bathymodiolinae).

Bathymodiolinae are giant mussels that were discovered at hydrothermal vents and harboring chemosynthetic symbionts. Due to their close phylogenetic relationship with seep species and tiny mussels from organic substrates, it was hypothesized that they gradually evolved from shallow to deeper environments, and specialized in decaying organic remains, then in seeps, and finally colonized deep-sea vents. Here, we present a multigene phylogeny that reveals that most of the genera are polyphyletic and/or paraphyletic. The robustness of the phylogeny allows us to revise the genus-level classification. Organic remains are robustly supported as the ancestral habitat for Bathymodiolinae. However, rather than a single step toward colonization of vents and seeps, recurrent habitat shifts from organic substrates to vents and seeps occurred during evolution, and never the reverse. This new phylogenetic framework challenges the gradualist scenarios "from shallow to deep." Mussels from organic remains tolerate a large range of ecological conditions and display a spectacular species diversity contrary to vent mussels, although such habitats are yet underexplored compared to vents and seeps. Overall, our data suggest that for deep-sea mussels, the high specialization to vent habitats provides ecological success in this harsh habitat but also brings the lineage to a kind of evolutionary dead end.

On the importance of oxidative folding in the evolution of conotoxins: cysteine codon preservation through gene duplication and adaptation.

Conotoxin genes are among the most rapidly evolving genes currently known; however, despite the well-established hypervariability of the intercysteine loops, the cysteines demonstrate significant conservation, with a site-specific codon bias for each cysteine in a family of conotoxins. Herein we present a novel rationale behind the codon-level conservation of the cysteines that comprise the disulfide scaffold. We analyze cysteine codon conservation using an internal reference and phylogenetic tools; our results suggest that the established codon conservation can be explained as the result of selective pressures linked to the production efficiency and folding of conotoxins, driving the conservation of cysteine at the amino-acid level. The preservation of cysteine has resulted in maintenance of the ancestral codon in most of the daughter lineages, despite the hypervariability of adjacent residues. We propose that the selective pressures acting on the venom components of cone snails involve an interplay of biosynthetic efficiency, activity at the target receptor and the importance of that activity to effective prey immobilization. Functional redundancy in the venom can thus serve as a buffer for the energy expenditure of venom production.

A tale of two chitons: is habitat specialisation linked to distinct associated bacterial communities?

Although most chitons (Mollusca: Polyplacophora) are shallow-water molluscs, diverse species also occur in deep-sea habitats. We investigated the feeding strategies of two species, Leptochiton boucheti and Nierstraszella lineata, recovered on sunken wood sampled in the western Pacific, close to the Vanuatu Islands. The two species display distinctly different associations with bacterial partners. Leptochiton boucheti harbours Mollicutes in regions of its gut epithelium and has no abundant bacterium associated with its gill. Nierstraszella lineata displays no dense gut-associated bacteria, but harbours bacterial filaments attached to its gill epithelium, related to the Deltaproteobacteria symbionts found in gills of the wood-eating limpet Pectinodonta sp. Stable carbon and nitrogen isotope signatures and an absence of cellulolytic activity give evidence against a direct wood-feeding diet; both species are secondary consumers within the wood food web. We suggest that the distinct associations with bacterial partners are linked to niche specialisations of the two species. Nierstraszella lineata is in a taxonomic family restricted to sunken wood and is possibly adapted to more anoxic conditions thanks to its gill-associated bacteria. Leptochiton boucheti is phylogenetically more proximate to an ancestral form not specialised on wood and may itself be more of a generalist; this observation is congruent with its association with Mollicutes, a bacterial clade comprising gut-associated bacteria occurring in several metazoan phyla.

The Terebridae and teretoxins: Combining phylogeny and anatomy for concerted discovery of bioactive compounds.

The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analyses of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationships within the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource.

Evolution of the Toxoglossa venom apparatus as inferred by molecular phylogeny of the Terebridae.

Toxoglossate marine gastropods, traditionally assigned to the families Conidae, Terebridae, and Turridae, are one of the most populous animal groups that use venom to capture their prey. These marine animals are generally characterized by a venom apparatus that consists of a muscular venom bulb and a tubular venom gland. The toxoglossan radula, often compared with a hypodermic needle for its use as a conduit to inject toxins into prey, is considered a major anatomical breakthrough that assisted in the successful initial radiation of these animals in the Cretaceous and early Tertiary. The pharmacological success of toxins from cone snails has made this group a star among biochemists and neuroscientists, but very little is known about toxins from the other Toxoglossa, and the phylogeny of these families is largely in doubt. Here we report the first molecular phylogeny for the Terebridae and use the results to infer the evolution of the venom apparatus for this group. Our findings indicate that most of the genera of terebrids are polyphyletic, and one species ("Terebra" (s.l.) jungi) is the sister group to all other terebrids. Molecular analyses combined with mapping of venom apparatus morphology indicate that the Terebridae have lost the venom apparatus at least twice during their evolution. Species in the genera Terebra and Hastula have the typical venom apparatus found in most toxoglossate gastropods, but all other terebrid species do not. For venomous organisms, the dual analysis of molecular phylogeny and toxin function is an instructive combination for unraveling the larger questions of phylogeny and speciation. The results presented here suggest a paradigm shift in the current understanding of terebrid evolution, while presenting a road map for discovering novel terebrid toxins, a largely unexplored resource for biomedical research and potential therapeutic drug development.