Hypobranchial Gland Research Articles

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.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1β1γ2 and α4β3γ2 receptors (IC50 1.5 and 1.0 μM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

Mutagenic impact of the biotoxin of veined rapa whelk <i>Rapana venosa </i> (Valenciennes, 1846) (Gastropoda, Muricidae)

The invasion of the predatory gastropod mollusk Rapana venosa, known as veined rapa whelk, in the Black Sea and its outspread in many regions of the World Ocean have led to complex structural changes in the communities of bivalve mollusks. When capturing a prey, the adult rapa whelk excretes a biotoxin from the hypobranchial gland that causes severe paralysis of the musculature of bivalve mollusks. Under experimental conditions, it was established that the biotoxin, after the short-term exposure of the fertilized eggs of the mussels Mytilus galloprovincialis to it, has a mutagenic effect, causing chromosomal aberrations in the embryos. The acentric groups of chromosomes, single and multiple chromosome bridges, chromosome lags and polyploidy indicate transformation or destruction of threads of the achromatin spindle in the mitosis anaphase. The chromosome fragmentation and emergence of ring chromosomes in the metaphase and anaphase of mitosis of mussel embryos are the consequence of the partial destruction of chromatin. The underdeveloped shell, anomalous development of D-veligers’ velum, and the changed form of the ‘eye spots’ and hepatopancreas of the larvae at the veliconcha stage result from chromosomal mutations during the embryo development. The mechanism of the rapa whelk biotoxin action on the chromosomal apparatus of mussel embryos is discussed.

The anatomy of the miniature predator Trigonulina ornata d’Orbigny, 1853 (Bivalvia: Anomalodesmata: Verticordiidae) from continental shelf waters off Brazil

The anatomy of one of the smallest non-septate carnivorous bivalves is described herein and is the first anatomical study of a member of the genus Trigonulina. This paper shows that similar to other verticordiids, Trigonulina ornata d’Orbigny, 1853 possesses a suite of anatomical features that include a prey-capturing inhalant siphon, a septum, an absence of labial palps and capacious mouth, which characterize it as a carnivorous marine bivalve. A discussion about the origin of the septibranch septum is provided showing that the condition seen in T. ornata could be representative of the transition within the Anomalodesmata between suspension-feeding type E ctenidia and the non-filamentar septibranch condition. Finally, fertilized and encapsulated eggs within the visceral mass, the presence of hypobranchial glands plus larval prodissoconch size collectively suggest self-fertilization and the possible early brooding behaviour by T. ornata.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.

Histology Atlas and Systematic Approach to Postmortem Examination of the Queen Conch Lobatus gigas

The queen conch Lobatus gigas is the second largest commercial fishery in the Caribbean, and overharvesting has resulted in significant population declines. Depleted populations are at greater risk of stochastic events, including disease epidemics; however, disease diagnosis in L. gigas has been limited by the lack of standard procedure and histological reference material. This manual outlines a systematic technique for postmortemexamination and compiles a comprehensive histology atlas to facilitate research regarding the pathology of L. gigas. Methods for euthanasia, dissection, sampling, and fixation are described, which produced optimal presentation and preservation of tissues. The recommended approach includes anesthesia with magnesium sulphate, extraction from shell, and euthanasia by incision through ganglia posterior to the buccal mass, followed by exposure of all tissues using four incisions. Anatomy is described for both sexes and standard sample locations are established. Tissue fixation is optimal using Davidson's formula. A histological reference for 16 tissues, nine of which were described for the first time in this species, is presented, including the anus, columellar muscle, digestive gland, esophagus, foot, ganglia and nerves, gill, gonad, heart, hypobranchial gland, kidney, mantle, nephridial gland, osphradium, small intestine and rectum. This manual is the first necropsy guide and histology atlas for L. gigas, a baseline resource for researchers monitoring health and disease in the species.

Brominated indoles from a marine mollusc inhibit inflammation in a murine model of acute lung injury.

New drug leads for the treatment of inflammation are urgently needed. Marine molluscs are widely used as traditional medicines for the treatment of inflammation. Here we report the positive effects of a hypobranchial gland (HBG) extract and the dominant bioactive compound 6-bromoisatin from the Muricidae mollusc Dicathais orbita, for reducing lipopolysaccharide (LPS) induced acute lung inflammation in a mouse model. Both 6-bromoisatin and the HBG extract suppressed the inflammatory response in mice that were pre-treated by oral gavage at 48, 24 and 1 h prior to LPS infusion. The inflammatory antagonists were tested at concentrations of 0.5 mg/g and 0.1 mg/g HBG extract and 0.1 mg/g and 0.05 mg/g 6-bromoisatin in carrier oil and all treatments reduced inflammation as indicated by a significant suppression of inflammatory markers present in bronchoalveolar lavage fluid (BALF), in comparison to LPS induced positive control mice administered the carrier oil alone (p < 0.0001). Tumour necrosis factor-alpha (TNFα) and interleukin-1 beta (IL-1β) levels, in addition to total protein concentration were all significantly reduced in BALF from mice treated with the extract or 6-bromoisatin. Furthermore, all treatment groups showed significant reductions in neutrophil sequestration and preservation of the lung tissue architecture compared to the positive control (p < 0.0001). The combined results from this study and our previous in vitro studies indicate that 6-bromoisatin in the HGB extracts inhibit the activation of inflammatory signalling pathway. The results from this study further confirm that the HBG extract from Muricidae molluscs and 6-bromoisatin are bioavailable and effective in vivo, thus have potential for development as natural therapeutic agents for inflammation.

A New Protein Extract Inhibitor from Hypobranchial Purple Gland of Hexaplex trunculus, a Mediterranean Mollusk, Impairs the Motility of Human Glioblastoma U87 and the HeLa Cell Line of Cervical Carcinoma Cells

ABSTRACTThe aim of this study is to evaluate the effect of hypobranchial gland protein extracts (HGPEs) of Hexaplex trunculus on the viability, cell adhesion, and migration of human U87 glioblastoma cells and the HeLa cell line obtained from epithelial cervical carcinoma cells. Analysis of the HGPE on polyacrylamide gel (12%) shows a variety of proteins whose molecular weights vary between 12 and 1OO kDa. Chromatographic analysis shows 16 peaks obtained at various retention times. Cytotoxic effect was observed after 24 hours of incubation at the concentrations 20, 40, and 60 μg/ml in a dose-dependent manner. Concentrations giving 50% inhibition (IC50) are 22 μg/ml for U87 and 15 μg/ml for HeLa cells. Our results show inhibition of U87 and HeLa cancer cell adhesion at concentrations of 10 and 20 µg/ml, respectively. High-pressure liquid chromatography fractions did not show antiadhesive effect on both cancer cell lines. The presence of HGPEs completely blocked the migration of the two cancer cell lines at 10 µg/ml. This inhibition is dose-dependent. IC50 is about 2.5 μg/ml for both cancer cells. The HGPE of Hexaplex trunculus may have the potential to serve as a model for future anticancer drug development with probably a synergistic activity of the proteins of this extract.

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.

Attenuation of UV-B exposure-induced inflammation by abalone hypobranchial gland and gill extracts

Exposure to solar ultraviolet B (UV-B) is a known causative factor for many skin complications such as wrinkles, black spots, shedding and inflammation. Within the wavelengths 280–320 nm, UV-B can penetrate to the epidermal level. This investigation aimed to test whether extracts from the tropical abalone [Haliotis asinina (H. asinina)] mucus-secreting tissues, the hypobranchial gland (HBG) and gills, were able to attenuate the inflammatory process, using the human keratinocyte HaCaT cell line. Cytotoxicity of abalone tissue extracts was determined using an AlamarBlue viability assay. Results showed that HaCaT cells could survive when incubated in crude HBG and gill extracts at concentrations between <11.8 and <16.9 μg/ml, respectively. Subsequently, cell viability was compared between cultured HaCaT cells exposed to serial doses of UV-B from 1 to 11 (x10) mJ/cm2 and containing 4 different concentrations of abalone extract from both the HBG and gill (0, 0.1, 2.5, 5 μg/ml). A significant increase in cell viability was observed (P<0.001) following treatment with 2.5 and 5 μg/ml extract. Without extract, cell viability was significantly reduced upon exposure to UV-B at 4 mJ/cm2. Three morphological changes were observed in HaCaT cells following UV-B exposure, including i) condensation of cytoplasm; ii) shrunken cells and plasma membrane bubbling; and iii) condensation of chromatin material. A calcein AM-propidium iodide live-dead assay showed that cells could survive cytoplasmic condensation, yet cell death occurred when damage also included membrane bubbling and chromatin changes. Western blot analysis of HaCaT cell COX-2, p38, phospho-p38, SPK/JNK and phospho-SPK/JNK following exposure to >2.5 μg/ml extract showed a significant decrease in intensity for COX-2, phospho-p38 and phospho-SPK/JNK. The present study demonstrated that abalone extracts from the HGB and gill can attenuate inflammatory proteins triggered by UV-B. Hence, the contents of abalone extract, including cellmetabolites and peptides, may provide new agents for skin anti-inflammation, preventing damage due to UV-B.

Extraction and Quantification of Bioactive Tyrian Purple Precursors: A Comparative and Validation Study from the Hypobranchial Gland of a Muricid Dicathais orbita.

Muricidae are marine molluscs known for the production of Tyrian purple and bioactive precursor compounds. A validation study for the extraction and analysis of secondary metabolites found in the hypobranchial gland of the muricid Dicathais orbita is reported, using high performance liquid chromatography–mass spectrometry (HPLC-MS) with diode array detector (DAD). Quantification of the dominant secondary metabolites from D. orbita is described, followed by a comparison of solvent extraction procedures and stability studies. The intra- and inter-day relative standard deviation (RSD) for tyrindoxyl sulphate was 0.46% and 0.17%, respectively. The quantification was linear for standards murexine, 6-bromoisatin, and tyrindoxyl sulphate. The limits of detection were 0.03, 0.004, and 0.07 mg/mL, respectively, and the limits of quantification were 0.09, 0.01, and 0.22 mg/mL, respectively. The results showed that alcoholic solvents were better for extracting choline ester and indoxyl sulphate ultimate precursors, while chloroform was more suitable for the extraction of the intermediate precursors. Multivariate analysis revealed significant differences in extract composition according to the solvent used. Stability testing showed an increase of the oxidative compounds 6-bromoisatin and putative tyrindoxyl S-oxide sulphate in the ethanol extracts while more degradation products were seen in the chloroform extracts after months of cold storage. The validated method was found to be simple, reproducible, precise, and suitable for quantification of the secondary metabolites of muricid molluscs for dye precursor and nutraceutical quality control, as well as applications in marine chemical ecology.

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.

Determination of Tyrian purple by high performance liquid chromatography with diode array detection

Indigotin, indirubin, 6-bromoindigotin, 6′-bromoindirubin, 6-bromoindirubin, 6,6′-dibromoindigotin and 6,6′-dibromoindirubin, the colouring components of Tyrian purple, are quantified by an efficient HPLC method coupled to a diode array detector. The compounds were separated using gradient elution, on a RP-column (Alltima C18, 250mm×3.0mm i.d., 5μm), thermostated at 35°C, with a mobile phase consisting of solvents (Α) H2O+0.1% (v/v) trifluoroacetic acid and (Β) acetonitrile+0.1% (v/v) trifluoroacetic acid, at a flow rate of 0.5mLmin−1. The method was validated in terms of linearity, detection and quantification limits, precision, accuracy, ruggedness and robustness, the latter with respect to small changes in column temperature and in flow-rate, pH- and solvent composition of the mobile phase. Under optimal conditions, the developed analytical scheme offers limits of detection in the range 0.02–0.05μgmL−1 and satisfactory linearity up to 2.5μgmL−1 for all analytes. Four samples produced from the hypobranchial glands of Hexaplex (Murex) trunculus molluscs, collected in the coastlines of Tunisia and Croatia, were treated with hot DMSO and analysed by the established HPLC method, using the standards addition approach. To evaluate the matrix effect, a comparison of the slopes of the standards in solvent regression curves with those of the standard addition method’s calibration curves, using the Student’s t-test was carried out. The accuracy was evaluated by recovery experiments. Amounts of indigotin, indirubin, and their mono- and dibrominated derivatives ranging between 0.01 to 12.2μgmg−1 were found in the DMSO extracts of the four molluscan samples.

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod.

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.

Mass spectrometry imaging reveals new biological roles for choline esters and Tyrian purple precursors in muricid molluscs.

Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown. One such example is the association between choline esters and Tyrian purple precursors in muricid molluscs. Mass spectrometry imaging (MSI) on nano-structured surfaces has emerged as a sophisticated platform for spatial analysis of low molecular mass metabolites in heterogeneous tissues, ideal for low abundant secondary metabolites. Here we applied desorption-ionisation on porous silicon (DIOS) to examine in situ changes in biodistribution over the reproductive cycle. DIOS-MSI showed muscle-relaxing choline ester murexine to co-localise with tyrindoxyl sulfate in the biosynthetic hypobranchial glands. But during egg-laying, murexine was transferred to the capsule gland, and then to the egg capsules, where chemical ripening resulted in Tyrian purple formation. Murexine was found to tranquilise the larvae and may relax the reproductive tract. This study shows that DIOS-MSI is a powerful tool that can provide new insights into marine chemo-ecology.

Detection and some properties of a high molecular weight toxin in the hypobranchial gland of strawberry conch Strombus luhuanus.

The extract from the hypobranchial gland of strawberry conch Strombus luhuanus was found to be lethal to mice. There were no marked regional and seasonal variations in toxicity although a considerable individual variation was recognized. The toxin was thermostable and extractable with aqueous solvents but not with organic solvents. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance of 400-500 k.

Are the Traditional Medical Uses of Muricidae Molluscs Substantiated by Their Pharmacological Properties and Bioactive Compounds?

Marine molluscs from the family Muricidae hold great potential for development as a source of therapeutically useful compounds. Traditionally known for the production of the ancient dye Tyrian purple, these molluscs also form the basis of some rare traditional medicines that have been used for thousands of years. Whilst these traditional and alternative medicines have not been chemically analysed or tested for efficacy in controlled clinical trials, a significant amount of independent research has documented the biological activity of extracts and compounds from these snails. In particular, Muricidae produce a suite of brominated indoles with anti-inflammatory, anti-cancer and steroidogenic activity, as well as choline esters with muscle-relaxing and pain relieving properties. These compounds could explain some of the traditional uses in wound healing, stomach pain and menstrual problems. However, the principle source of bioactive compounds is from the hypobranchial gland, whilst the shell and operculum are the main source used in most traditional remedies. Thus further research is required to understand this discrepancy and to optimise a quality controlled natural medicine from Muricidae.

Vibrio harveyi adheres to and penetrates tissues of the European abalone Haliotis tuberculata within the first hours of contact.

Vibrio harveyi is a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abalone Haliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection of V. harveyi in the European abalone. The results indicate that the duration of contact between V. harveyi and the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized by V. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain of V. harveyi in abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact. V. harveyi was also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such as V. harveyi in mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration of V. harveyi in European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry of V. harveyi in the European abalone is the area situated between the gills and the hypobranchial gland.

Supercritical CO2 extraction of bioactive Tyrian purple precursors from the hypobranchial gland of a marine gastropod

Supercritical CO2 provides considerable advantages over traditional solvents for the extraction of bioactive compounds from organic matter. Here we demonstrate the use of supercritical CO2 as an efficient and safe alternative to traditional solvent extraction for the recovery of bioactive Tyrian purple precursors tyrindoleninone, 6-bromoisatin and tyriverdin from the marine mollusc Dicathais orbita. The effect of pressure on the selective extraction of brominated indoles was tested at 15, 30 and 50MPa CO2, and was compared to traditional chloroform extract composition and yields. Extracts obtained from 15MPa selectively concentrated 6-bromoisatin, at 78% of the extract composition, whereas increased pressures of 30 and 50MPa increased the solvating power of supercritical CO2 to include the more lipophilic tyrindoleninone at 35 and 29% respectively, and tyriverdin at 23 and 40% respectively. This extraction method was also effective in separating the brominated indoles from toxic choline esters in the mollusc extracts. Extract yields from supercritical CO2 were comparable to solvent extraction relative to whole whelk weight. This provides a viable alternative for nutraceutical development that does not rely on the use of toxic solvents.

Comparative Study of Nutritional Values of Edible Viscera Mediterranean Mollusks Gastropods hexaplex trunculus and bolinus brandaris. Hypobranchial glands inhibit Human Glioblastoma U87 Tumor Cells Adhesion and Proliferation

This study aims first to identify nutritionals values of edible mix Muscle Hypobranchial Gland MHG, fresh and cooked extract from internal organs of two gastropods mollusks Hexaplex trunculus HT and Bolinus brandaris BB males and females. Second, to characterize and evaluate the role of hypobranchial glands extract HBGE of these mollusks gastropods on adhesion and cell proliferation of human glioblastoma tumor cells U87. Biochemical analyzes show that fresh MHG, HT and BB are rich in Water 82.52% in HT males, 79.23% in HT females. They are rich in Proteins 12.79 % in BB females to 10.30% in HT females) and in Energy value 79.14 kcal in BB females to 69.48kcal in HT males. They contain fewer carbohydrates 6.27% in BB males to 5.40% in HT males, less total ash 1.52% in HT females to 1.11% in HT males and less lipids 2.55% in HT females to 0.74% in BB males. Further, HBGE of HT inhibited U87 cell adhesion to fibrinogen at 15 mg/ml. This inhibition was accelerated at 20 mg/ml without exceeding 50% of inhibition. HBGE of BB reduced glioblastoma cells adhesion upon to 5 mg/ml with IC50 values of 9 mg/ml for male and 20 mg/ml for female. U87 cells treated with HBGE HT and BB decreased significantly cell proliferation by 50% and 70% respectively. Our results lead us to encourage the use of these marine snails, source of protein and high energy values. Moreover, HBGE may have the potential to serve as a template for future anticancer-drug development. Keywords : Mix Muscle- Hypobranchial Gland, Hexaplex Trunculus, Bolinus Brandaris, Nutritional Values, Glioblastoma U87 Cells, Cell Adhesion and Proliferation.