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.

First Report of the Molecular Mechanism of Resistance to Tribenuron-Methyl in Silene conoidea L.

Silene conoidea L. is an annual troublesome broadleaf weed in winter wheat fields in China. In recent years, field applications of tribenuron-methyl have been ineffective in controlling S. conoidea in Hebei Province, China. The aim of this study was to determine the molecular basis of tribenuron-methyl resistance in S. conoidea. Whole-plant response assays revealed that the resistant population (R) exhibited a higher level of resistance (382.3-fold) to tribenuron-methyl. The R population also showed high cross-resistance to other acetolactate synthase (ALS) inhibitors, including imazethapyr, bispyribac-sodium and florasulam. However, the R population could be controlled by the field-recommended rates of bentazone, MCPA, fluroxypyr, carfentrazone-ethyl and bromoxynil. In vitro ALS activity assays indicated that the tribenuron-methyl I50 value for the R population was 18.5 times higher than those for the susceptible population (S). ALS gene sequencing revealed an amino acid mutation, Trp-574-Leu, in the R population. Pretreatment with the P450 inhibitor malathion indicated that the R population might have cytochrome P450-mediated metabolic resistance. These results suggest that the Trp-574-Leu mutation and P450-mediated enhanced metabolism coexist in S. conoidea to generate tribenuron-methyl resistance. This is the first time that target-site and non-target-site resistance to tribenuron-methyl has been reported in S. conoidea.

The complete mitogenome of Phymorhynchus sp. (Neogastropoda, Conoidea, Raphitomidae) provides insights into the deep-sea adaptive evolution of Conoidea.

The deep-sea environment is characterized by darkness, hypoxia, and high hydrostatic pressure. Mitochondria play a vital role in energy metabolism; thus, they may endure the selection process during the adaptive evolution of deep-sea organisms. In the present study, the mitogenome of Phymorhynchus sp. from the Haima methane seep was completely assembled and characterized. This mitogenome is 16,681 bp in length and contains 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. The gene order and orientation were identical to those of most sequenced conoidean gastropods. Some special elements, such as tandem repeat sequences and AT-rich sequences, which are involved in the regulation of the replication and transcription of the mitogenome, were observed in the control region. Phylogenetic analysis revealed that Conoidea is divided into two separate clades with high nodal support. Positive selection analysis revealed evidence of adaptive changes in the mitogenomes of deep-sea conoidean gastropods. Eight residues located in atp6, cox1, cytb, nad1, nad4, and nad5 were determined to have undergone positive selection. This study explores the adaptive evolution of deep-sea conoidean gastropods and provides valuable clues at the mitochondrial level regarding the exceptional adaptive ability of organisms in deep-sea environments.

Transcriptomic Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus.

Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.

Phylogenetic classification and generic delineation of Calyptosphaeria gen. nov., Lentomitella, Spadicoides and Torrentispora (Sordariomycetes).

The genus Ceratostomella has a long history of taxonomic confusion. While species with evanescent asci have been transferred to the Microascales and Ophiostomatales, the taxonomic status of species with persistent asci has not been completely resolved. In previous studies using DNA sequence data, cultures and morphology, several Ceratostomella spp. were allocated in 13 genera in the Eurotiomycetes and Sordariomycetes. In our study, the systematics of the remaining Ceratostomella spp. with persistent asci is revisited with new collection data, cultures and phylogeny based on novel DNA sequences from six nuclear loci. Bayesian inference and Maximum Likelihood analyses support the monophyly of several wood-inhabiting species formerly classified in Ceratostomella and other unknown morphologically similar taxa and their division into four genera, i.e. Lentomitella, Spadicoides, Torrentispora and the newly described Calyptosphaeria. This robust clade represents the order Xenospadicoidales in the Sordariomycetidae. Comparative analysis of the ITS2 secondary structure revealed a genetic variation among Lentomitella isolates; 11 species were recognised, of which five are newly introduced and two are new combinations. Other taxonomic novelties include four new species and eight new combinations in Calyptosphaeria, Spadicoides, and Torrentispora. Molecular data suggest that Spadicoides is polyphyletic. The core of the genus is positioned in the Xenospadicoidales; Spadicoides s. str. is experimentally linked with sexual morphs for the first time. Based on DNA sequence data, the monotypic genera Xenospadicoides and Pseudodiplococcium are reduced to synonymy under Spadicoides, while Fusoidispora and Pseudoannulatascus are synonymised with Torrentispora. Members of the Xenospadicoidales inhabit decaying wood in terrestrial and freshwater environments and share a few morphological characters such as the absence of stromatic tissue, ascomata with a cylindrical or rostrate neck, similar anatomies of the ascomatal walls, thin-walled unitunicate asci with a non-amyloid apical annulus, disintegrating paraphyses, usually ellipsoidal to fusiform ascospores and holoblastic-denticulate or tretic conidiogenesis. Revised Ceratostomella spp. with persistent asci are listed and the taxonomic status of each species is re-evaluated based on revision of the holotype and other representative material, published details and available phylogenetic data.

From common to rare Zingiberaceae plants - A metabolomics study using GC-MS.

Zingiberaceae plants, commonly known as gingers, have been popular for their medicinal and culinary uses since time immemorial. In spite of their numerous health-promoting applications, many Zingiberaceae plants still receive no scientific attention. Moreover, existing reports mostly focused only on the Zingiberaceae rhizomes. Here, untargeted metabolite profiling using Gas Chromatography - Mass Spectrometry (GC-MS) was used to compare the metabolic composition of leaves and rhizomes of the more common gingers, Zingiber officinale Rosc. (ZO), Curcuma longa L. (CL), and Etlingera elatior (Jack) R.M. Smith (EE), and the rare gingers, Amomum muricarpum Elmer (AM), Etlingera philippinensis (Ridl.) R.M. Smith (EP), and Hornstedtia conoidea Ridl. (HC). Principal component analysis (PCA) demonstrated that different species show substantial chemical differentiation and revealed potential markers among the different Zingiberaceae plants. Interestingly, the leaves of AM, CL, EE, EP, and HC had significantly higher levels of chlorogenic acid than ZO. Moreover, rhizomes of EP and HC were found to contain significantly higher levels of amino acids than ZO. Sugars and organic acids were generally less abundant in ZO leaves and rhizomes than in the other gingers. The leaves of EP and rhizomes of AM were found most similar to the leaves and rhizomes of common gingers, respectively. Results of this study provide significant baseline information on assessing the possible usage of the leaves of common gingers and further propagation and exploration of EP and AM. This study, being the first metabolomics report on rare plants such as AM, EP and HC, affirms the usefulness of untargeted metabolite profiling in exploring under-investigated plants.

On the oviposition and egg masses of Conus regius (Neogastropoda: Conidae) from northeastern Brazil / Sobre a oviposição e massas de ovos de Conus regius (Neogastropoda: Conidae) no nordeste do Brasil

Abstract Characteristics of the egg masses of Conus regius Gmelin, 1791 are described and figured for the first time for the coast of Brazil based on a female specimen found in the process of oviposition during the day in the state of Bahia, northeastern Brazil. Two clusters of egg masses were found in the subtidal zone of Itapuã beach attached to rocky substrate in a completely unprotected site. Oviposition likely began at least one day earlier, since the specimen had already affixed an entire egg mass and was ovipositing a second cluster at the time it was found. The egg masses were arranged in short, irregular rows of three to nine closely spaced capsules in parallel and facing the same direction. One egg mass cluster consisted of 34 capsules. Conus regius capsules are semi-transparent, vasiform in side view, higher than broad and have flattened to slightly convex sides with slight wrinkles constituted by transversal ridges. Conus regius is on the IUCN Red List of Threatened Species, but is still intensively collected in the study area and surrounding coastal environment by fishermen for the purposes of selling shells and as a food source.

Resumo Características de massas de ovos de Conus regius Gmelin, 1791 são descritas e figuradas pela primeira vez para a costa do Brasil com base em um espécime encontrado em processo de oviposição, durante o dia, no estado da Bahia, nordeste do Brasil. Dois aglomerados de massa de ovos foram achadas na zona subtidal da praia de Itapuã fixa a substrato rochoso em uma área completamente desprotegida. O processo de oviposição, provavelmente, começou um dia antes, uma vez que o espécime já tinha afixado uma massa de ovos e estava depositando um segundo aglomerado de ovos no momento em que foi encontrado. As massas de ovos apresentaram arranjos em fileiras curtas e irregulares contendo três a nove cápsulas estreitamente espaçadas, paralelas e posicionadas na mesma direção. A primeira massa de ovos observada conteve um total de 34 cápsulas. As cápsulas de Conus regius são semitransparentes, vasiformes em vista lateral (mais altas do que largas) e possuem parte lateral suavemente convexa a achatada com rugas suaves constituídas por elevações transversais. Conus regius está na Lista Vermelha de Espécies Ameaçadas da IUCN, mas ainda é intensamente coletada na área de estudo e ambientes costeiros circunvizinhos por pescadores por conta da venda das conchas e como fonte alimentar.

Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata.

Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C-C-CC-C-C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides.

Characterization of the complete mitochondrial genome of Conus tribblei Walls, 1977.

The genus Conus sensu lato consists of 500-700 species. However, the mitochondrial genomes of only few species have been fully sequenced and reported so far. In this study, the complete mitochondrial genome of Conus tribblei, a member of the poorly known subgenus Splinoconus is sequenced with the mean coverage of 604×. The mitochondrial genome is 15 570 bp long and consists of genes encoding for 13 respiratory chain proteins, 22 tRNA and 2 rRNA. The gene organization is highly conserved among the Conus species. The longest intergenic region between tRNA-Phe and cytochrome c oxidase subunit III (cox3), which in C. tribblei is 169 bp long and contains a 112 bp long segment of inverted repeat, represents the putative control region. The control regions of Conus species exhibited variability in the length and position of the inverted repeats. Therefore, this region may have the potential to be used as a genetic marker for species discrimination.

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 venom peptides, 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 homology-based searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, such as actinoporin, commonly found in venoms. This research provides novel insights into venom evolution and recruitment 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.

Biodiversity of cone snails and other venomous marine gastropods: evolutionary success through neuropharmacology.

Venomous marine snails (superfamily Conoidea) are a remarkably biodiverse marine invertebrate lineage (featuring more than 10,000 species). Conoideans use complex venoms (up to 100 different components for each species) to capture prey and for other biotic interactions. Molecular phylogeny and venom peptide characterization provide an unusual multidisciplinary view of conoidean biodiversity at several taxonomic levels. Venom peptides diverge between species at an unprecedented rate through hypermutation within gene families. Clade divergence within a genus occurs without recruiting new gene families when a saltatory event, such as colonization of new prey types (e.g., fish), leads to a new radiation. Divergence between genera in the same family involves substantial divergence in gene families. In the superfamily Conoidea, the family groups recruited distinct sets of different venom gene superfamilies. The associated morphological, behavioral, and prey-preference changes that accompany these molecular changes are unknown for most conoidean lineages, except for one genus, Conus, for which many associated phenotypic changes have been documented.

Pharmacological characterization of venoms obtained from Mexican toxoglossate gastropods on isolated guinea pig ileum

The protein-containing extracts prepared from the venom ducts of Conus austini, Conus spurius and Polystira albida caused a concentration-dependent inhibition of spontaneous contractions in guinea pig ileum. The most potent extract was obtained from P. albida venom ducts (IC50 equal 0.11 more or less 0.02 microg protein/mL). The three extracts produced a moderate inhibition of contractions elicited by acetylcholine (ACh 1 microM), suggesting the presence of anticholinergic compounds. The contractile response elicited by nicotine (10 microM) was significantly reduced by the extracts prepared from the ducts of C. austini and P. albida, which indicates that the venom produced by these species contains toxins that target neuronal nicotinic receptors. All three extracts significantly inhibited contractions evoked by histamine (0.5 miM), particularly those from C. spurius and P. albida. These findings reveal the presence of antihistaminergic compounds not previously described in any conoidean venom. Finally, we found that only the extract prepared from C. spurius ducts decreased KCl (60 mM)-induced contractions, indicating that the venom of this snail contains compounds that block voltage-dependent Ca2 more or Na more channels.

Estudio Histológico de Boca, Esófago, Estómago e Intestino de Polystira albida (Gastropoda: Turridae) / Histology Study of Mouth, Esophagus, Stomach and Intestine of Polystira albida (Gastropoda: Turridae)

En la presente investigación se realizó la caracterización histológica del tubo digestivo de Polystira albida (Toxoglossa:Turridae) mediante microscopía óptica, utilizando ejemplares colectados en la Sonda de Campeche. Los resultados del análisis muestran que los labios, la boca y la probóscide presentan un epitelio cilindrico simple ciliado. La cavidad bucal tiene un epitelio cilindrico estratificado. El esófago presenta un epitelio cilindrico simple con células glandulares granulares acidófilas (naturaleza proteica), y es el sitio donde comienza la digestión. El estómago se divide en tres regiones histológicas, y tiene dos tipos de epitelio, uno cilindrico simple ciliado y otro cilindrico simple; el primero está formado por abundantes células glandulares acidófilas y basófilas (naturaleza proteica y glicoproteica', respectivamente); y el segundo contiene slo células glandulares acidófilas. La glándula digestiva está constituida por adenómeros túbulo-acinosos con células glandulares acidófilas y basófilas, en diferentes grados de secreción y los conductos presentan un epitelio cilindrico ciliado con células glandulares de ambos tipos. El intestino se divide en cinco regiones histológicas, la primera presenta un tiflosol con dos tipos de epitelio, cilindrico simple y cilindrico simple con chapa estriada; y el resto del tracto tiene un epitelio cilindrico simple ciliado con células glandulares granulares acidófilas y pliegues de diferentes tamaños que aumentan o reducen la cavidad del conducto. Se concluye que existen diferencias, principalmente en la región anterior del tubo digestivo (cavidad bucal, esófago y estómago), con respecto a las descripciones generales realizadas para la subclase Prosobranchia.

In the present investigation it was realized a histological characterization of the digestive tract of Polystira albida (Toxoglossa: Turridae), by using optic microscopy of specimens collected in Sonda de Campeche. Results of analysis show that lips, mouth and proboscis present a simple columnar epithelium with cilia. Buccal cavity has a stratified columnar epithelium. Esophagus presents a simple columnar epithelium with acidophilic granular glandular cells (proteic nature), and it is the place where digestion starts. Stomach is divided in three histological regions that have two epithelial types, simple columnar with cilia and without one; the first is formed by a lot of acidophilic and basophilic glandular cells (proteic and glycoproteic nature respectively), and the second only contains acidophilic glandular cells. Digestive gland is constituted by tubule-acinous adenomers with acidophilic and basophilic glandular cells in different grades of secretion, and ducts have simple columnar epithelium with cilia and scarcely glandular cells. Intestine is divided in five histological regions, the first presents atyphlosole with two types of epithelium, simple columnar with stratified border; the rest of the tract has simple columnar epithelium with cilia, acidophilic granular glandular cells and folds of different sizes that increase or reduce the cavity duct. Based on previous this, we conclude that in this study exist differences principally in front region of digestive tract (buccal cavity, esophagus and stomach) compared to general descriptions realized for Prosobranchia subclass.