Complete mitochondrial genome and phylogenetic analysis of Mancinella alouina.

BACKGROUND: The Muricidae family in the Class Gastropoda comprises numerous species with a vast range of morphological features and a worldwide presence. The phylogeny of the Muricidae has been analyzed in previous studies; however, the evolutionary relationships among the main branches of the Muricidae remain unknown. METHODS AND RESULTS: In the present study, the mitochondrial genome of Mancinella alouina was sequenced. The mitochondrial genome was found to be 16,671 bp in length and made up of 37 genes (13 protein-coding genes, 22 transfer RNA and 2 ribosomal RNA genes). The genome has an A-T-rich region (66.5% A + T content) and all of the PCGs use the ATN start codon and the TAG or TAA stop codons. The mitochondrial gene arrangement of Mancinella alouina is similar to that of other Muricidae, except for Ocinebrellus inornatus and Ceratostoma burnetti. On the basis of a flexible molecular clock model, time-calibrated phylogenetic results indicate that the genus Mancinella diverged roughly 18.09 Mya, and that the family Muricidae emerged in the Late Cretaceous. CONCLUSIONS: This study reveals the structural and sequence information features of the mitochondrial genome of Mancinella alouina. This study provides evidence for the relationships within the family Muricidae at the molecular level, and infer the divergence time. The results of phylogenetic analyses strongly support the current classification.

Hemocyanins of Muricidae: New 'Insights' Unravel an Additional Highly Hydrophilic 800 kDa Mass Within the Molecule.

Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-)decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins.

[Tetramine Contents in the Salivary Glands from 16 Species of Marine Carnivorous Gastropods Collected along Japanese Coasts].

Tetramine (tetramethyl ammonium ion), a neurotoxin, is present at high levels in the salivary glands of buccinid gastropods and is responsible for human intoxication due to consumption of the gastropods. We used LC-MS/MS to examine the tetramine contents of salivary glands from 16 species of carnivorous gastropods collected along Japanese coasts. Tetramine was detected in all specimens except for Babylonia japonica. High levels of tetramine were detected in whelks, Neptunea lamellosa (1,380-9,410 µg/g of salivary gland) and N. purpurea (1,190-7,400 µg/g of salivary gland). Although consumption of N. lamellosa is well-known cause of foodborne tetramine poisoning, it was newly discovered that N. purpurea has tetramine. In addition, we found 7 other species of gastropods containing tetramine: Siphonalia cassidariaeformis (117-135 µg/g), S. fusoides (204 µg/g), Buccinum inclytum (2.94-3.40 µg/g), and B. aniwanum (0.700 µg/g) of the family Buccinidae, and Fusinus perplexus (397 µg/g), F. ferrugineus (105 µg/g), and F. forceps salisburyi (67.5 µg/g) of the family Fasciolariidae. The present study, together with previous studies, shows that gastropods with salivary glands containing more than 1,000 µg tetramine/g of salivary gland, including the genus Neptunea as well as Fusitriton oregonesis and Hemifusus tuba, carry a high risk of tetramine poisoning, and their salivary glands should be removed before consumption to prevent food poisoning.

Anti-Inflammatory Activity and Structure-Activity Relationships of Brominated Indoles from a Marine Mollusc.

Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 µg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.

Transcriptome of the Australian Mollusc Dicathais orbita Provides Insights into the Biosynthesis of Indoles and Choline Esters.

Dicathais orbita is a mollusc of the Muricidae family and is well known for the production of the expensive dye Tyrian purple and its brominated precursors that have anticancer properties, in addition to choline esters with muscle-relaxing properties. However, the biosynthetic pathways that produce these secondary metabolites in D. orbita are not known. Illumina HiSeq 2000 transcriptome sequencing of hypobranchial glands, prostate glands, albumen glands, capsule glands, and mantle and foot tissues of D. orbita generated over 201 million high quality reads that were de novo assembled into 219,437 contigs. Annotation with reference to the Nr, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases identified candidate-coding regions in 76,152 of these contigs, with transcripts for many enzymes in various metabolic pathways associated with secondary metabolite biosynthesis represented. This study revealed that D. orbita expresses a number of genes associated with indole, sulfur and histidine metabolism pathways that are relevant to Tyrian purple precursor biosynthesis, and many of which were not found in the fully annotated genomes of three other molluscs in the KEGG database. However, there were no matches to known bromoperoxidase enzymes within the D. orbita transcripts. These transcriptome data provide a significant molecular resource for gastropod research in general and Tyrian purple producing Muricidae in particular.

Assessing the scale and ecological impact of derelict and discarded fishing gear across Thailand via the MARsCI citizen science protocol.

South-East Asia is among the least studied regions for the growing issue of marine debris pollution, despite being a major contributor towards global marine debris. In the present study, we provide the preliminary results from the MARsCI project, a survey protocol designed to utilise citizen science to facilitate data collection on the ecological impact of discarded fishing gear (DFG) in Thailand. Over a three-year period, 103 surveys were carried out across Thailand, resulting in impact assessment of 606 pieces of DFG. Our findings indicate corals are regularly impacted by DFG in Thai waters and that isolated marine habitats may be more severely impacted than near-shore sites. We further identify crabs, muricid snails, and demersal fish to be among the most regularly entangled animals. We discuss our findings in the context of earlier work from Thailand, and conduct a critical review of the protocol itself, identifying improvements for future efforts.

Three-dimensional visualization of predatory gastropod feeding teeth with synchrotron scanning.

Several families of neogastropod mollusks independently evolved the ability to drill through mineralized prey skeletons using their own mineralized feeding teeth, sometimes with shell-softening chemical agents produced by an organ in the foot. Teeth with more durable tooth shapes should extend their use and improve predator performance, but past studies have described only the cusped-side of teeth, mostly overlooking morphologies related to functional interactions between teeth. Here, we describe the three-dimensional morphology of the central drilling tooth (rachidian) from four species of the neogastropod family Muricidae using synchrotron tomographic microscopy and assemble a three-dimensional model of a multitooth series in drilling position for two of them to investigate their dynamic form. We find two new types of articulating surfaces, including a saddle joint at either end of the rachidian and a large tongue-and-groove joint in the center. The latter has a shape that maximizes contact surface area between teeth as they rotate away from each other during drilling. Articulating joints have not been described in Neogastropod radula previously, but they are consistent with an earlier hypothesis that impact forces on individual teeth during predatory drilling are dispersed by tooth-tooth interactions.

Recognising mollusc shell contours with enlarged spines: Wavelet vs Elliptic Fourier analyses.

Gastropod shells may present large spines and sharp shapes that vary according to environmental, taxonomic, and evolutionary factors. In these cases, classic morphometric methods used to study shell contour might not provide a clear representation of morphological shell based on angular decomposition of contour. The present study analyzed and compared for the first time the robustness of the contour analysis using wavelet transformed and Elliptic Fourier descriptors for gastropod shells including enlarged spines. For that, we analyzed two geographical and ecological separated populations of Bolinus brandaris from the NW Mediterranean Sea. Results showed that contour analysis of gastropod shells with enlarged spines can be analyzed using both methodologies, but the wavelet analysis provided a better local discrimination. From an ecological perspective, shells with spines of different sizes were observed in both localities suggesting a wide plasticity of the species.

An unreported bis-abeo cembrane-type diterpenoid with antioxidative and anti-lipoxygenase activities from the muricid gastropod mollusc Chicoreus ramosus.

The chemical analyses of ethyl acetate-methanol (EtOAc-MeOH) extract of muricid gastropod mollusk, Chicoreus ramosus from the southeastern coast of Indian peninsular led to the identification of unprecedented cembrane-type diterpenoid, which was characterized as (3E, 6E, 10E)-8a-butoxy-17(15→14), 20(12→11)-bis-abeo-cembra-3,6,10,14(17),15-pentaene (1). The structure of the studied cembrane was unambiguously assigned through the extensive spectroscopic experimentations. The antioxidant potentials of the bis-abeo cembrane as determined by in vitro 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiozoline-6-sulfonic acid diammonium salt radical quenching potentials were greater (IC50 < 0.40 mg/mL) related to α-tocopherol (IC50 > 0.60 mg/mL). The pro-inflammatory anti-5-lipoxygenase potential of studied cembrane was higher (IC50 < 0.80 mg/mL) related to those demonstrated by ibuprofen and sodium salicylate (IC50 > 0.90 mg/mL).

Antioxidant drimane-type sesquiterpenoid from muricid gastropod Chicoreus ramosus attenuates pro-inflammatory 5-lipoxygenase and carbolytic enzymes.

The muricid gastropod, Chicoreus ramosus, is a nutrient-enriched food source available along the coastal peninsular of the Indian subcontinent. This study was aimed at bioactivity-directed chromatographic fractionation of the organic extract of C. ramosus to isolate an unprecedented drimane-type sesquiterpenoid Ramosane, characterized as 3-hydroxy-7,9b-dimethyl-5-methylene-8-pentyl-octahydro-1H-cyclopenta[a]naphthalen-9(2H)-one. The compound possessed potential antioxidant activities {2, 2'-diphenyl-1-picryl hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activities of IC50 1.42 mM and 1.72 mM, respectively} and was proportionate with those (IC50 1.39 and 1.69 mM, respectively) exhibited by α-tocopherol. The studied sesquiterpenoid exhibited potential attenuation property countering the pro-inflammatory 5-lipoxygenase (IC50  ~ 4 mM), and its activity was analogous with that exhibited by the anti-inflammatory ibuprofen (IC50 4.36 mM), whereas its carbolytic α-amylase activity (IC50 0.96 mM) was commensurate with that displayed by acarbose (IC50 0.43 mM). The isolated metabolite might anticipate as potential naturally derived bioactive constituent in functional food and pharmaceutical applications. PRACTICAL APPLICATIONS: The edible marine muricid gastropod C. ramosus is a prominently available gastropod species of commercial significance in the coastal regions of Indian Peninsula. An unprecedented drimane-type sesquiterpenoid Ramosane was isolated through the bioactivity-directed chromatographic fractionation of the organic extract of muricidae C. ramosus displaying potential anti-inflammatory and anti-diabetic properties. The present study apprehended the prospective of drimane-type sesquiterpenoid derivative Ramosane purified from C. ramosus as a naturally derived pharmacophore with anti-diabetic and anti-inflammatory potential for utilization in functional food and pharmaceutical formulations to minimize the likelihood of inflammation and hyperglycemic pathologies.

The complete mitochondrial genome of Indothais lacera (Neogastropoda: Muricidae).

The rock shells Thais is the most important genera of Muricidae. However, the systemically classification and phylogenetic studies have so far been limited. In this study, we report the complete mitochondrial genome sequence of Indothais lacera. The mitogenome has 15,272 base pairs (68.1% A + T content) and made up of a total of 37 genes (13 protein-coding, 22 transfer RNAs and two ribosomal RNAs), but no control region. This study was the first available complete mitogenomes of Indothais and will provide useful genetic information for future phylogenetic and evolutionary classification of Thais.

Unanticipated discovery of two rare gastropod molluscs from recently located hydrothermally influenced areas in the Okinawa Trough.

BACKGROUND: The deep-sea hydrothermal vent is one of the most 'extreme' environments in the marine realm. Few species are capable of inhabiting such ecosystems, despite extremely high productivity there supported by microbial chemosynthesis, leading to high biomass and low species richness. Although gastropod molluscs are one of the main constituents of megafaunal communities at vent ecosystems, most species belong to several typical families (e.g., Provannidae, Peltospiridae, Lepetodrilidae) specialised and adapted to life at vents. METHODS: During recent surveys of Okinawa Trough hydrothermal vent systems, two snails atypical of vent ecosystems were unexpectedly found in newly discovered hydrothermally influenced areas. Shell and radular characteristics were used to identify the gastropods morphologically. RESULTS: One species was a vetigastropod, the calliostomatid Tristichotrochus ikukoae (Sakurai, 1994); and the other was a caenogastropod, the muricid Abyssotrophon soyoae (Okutani, 1959). Both gastropods were previously only known from regular non-chemosynthetic deep-sea and very rare-only two definitive published records exist for T. ikukoae and three for A. soyoae. The radula formula of Tristichotrochus ikukoae is accurately reported for the first time and based on that it is returned to genus Otukaia. For both species, barcode sequences of the cytochrome c oxidase I (COI) gene were obtained and deposited for future references. DISCUSSION: These new records represent the second record of calliostomatids from vents (third from chemosynthetic ecosystems) and the third record of muricids from vents (tenth from chemosynthetic ecosystems), and extend the distribution of both species to the southwest. Neither family has been recorded at chemosynthetic ecosystems in the western Pacific. Both were from weakly diffuse flow areas not subject to high temperature venting but were nevertheless associated with typical vent-reliant taxa such as Lamellibrachia tubeworms and Bathymodiolus mussels. These new records show that these species are capable of tolerating environmental stress associated with weak hydrothermally influenced areas, despite not being vent endemic species, adding to the list of known vent/non-vent species intersections. This signifies that such weakly influenced areas may provide key habitats for them, and that such areas may play a role in the evolution of biological adaptations to 'extreme' chemosynthetic ecosystems.

The complete mitochondrial genome of Rapana venosa (Gastropoda, Muricidae).

The complete mitochondrial (mt) genome of the veined rapa whelk, Rapana venosa, was determined using genome walking techniques in this study. The total length of the mt genome sequence of R. venosa was 15,271 bp, which is comparable to the reported Muricidae mitogenomes to date. It contained 13 protein-coding genes, 21 transfer RNA genes, and two ribosomal RNA genes. A bias towards a higher representation of nucleotides A and T (69%) was detected in the mt genome of R. venosa. A small number of non-coding nucleotides (302 bp) was detected, and the largest non-coding region was 74 bp in length.

The complete mitochondrial genome of Menathais tuberosa (Gastropoda, Neogastropoda, Muricidae) collected from Chuuk Lagoon.

Complete mitochondrial genome of the knobbed rock shell sea snail Menathais tuberosa (Röding, 1798) has been sequenced and phylogenetic relationships evaluated due to mitochondrial protein coding genes. The size of mitochondrial genome for M. tuberosa is 15,294 bp and the nucleotide composition of the mitochondrial genome is 28.4% A, 16.5% C, 17.6% G and 37.5% T. Reconstructed phylogenetic tree of the Neogastropoda showed that M. tuberosa is in the monophyletic Muricidae. This is the first record of complete mitochondrial genome from the genus.