Vibrio harveyi adheres to and penetrates tissues of the European abalone Haliotis tuberculata within the first hours of contact.
ABSTRACT: 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.
Project description:Ostracod crustaceans are diverse and ubiquitous in aqueous environments today but relatively few known species have gills. Ostracods are the most abundant fossil arthropods but examples of soft-part preservation, especially of gills, are exceptionally rare. A new ostracod, Spiricopia aurita (Myodocopa), from the marine Silurian Herefordshire Lagerstätte (430 Mya), UK, preserves appendages, lateral eyes and gills. The respiratory system includes five pairs of gill lamellae with hypobranchial and epibranchial canals that conveyed haemolymph. A heart and associated vessels had likely evolved in ostracods by the Mid-Silurian.
Project description: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.
Project description: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.
Project description: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 ? (TNF?) 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 ?g/mL, respectively. The hypobranchial gland extract also inhibited the production of TNF? 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, TNF?, 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 (NF?B) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NF?B 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 NF?B, 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.
Project description: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.
Project description:Withering syndrome (WS), an infectious disease caused by intracellular bacteria Candidatus Xenohaliotis californiensis, has provoked significant economic losses in abalone aquaculture. The pathogen infects gastroenteric epithelia, including digestive gland, disrupting the digestive system and causing a progressive wilting in abalone. Nonetheless, our knowledge about WS implications in digestive gland microbiota, and its role in diseases progress remains largely unknown. This study aims to determine whether digestive gland-associated microbiota differs between healthy red abalone (Haliotis rufescens) and red abalone affected with WS. Using high-throughput sequencing of the V4 region of the 16S rRNA gene, our results revealed differences in microbiota between groups. Bacterial genera, including Mycoplasma, Lactobacillus, Cocleimonas and Tateyamaria were significantly more abundant in healthy abalones, whilst Candidatus Xenohaliotis californiensis and Marinomonas were more abundant in WS-affected abalones. Whilst Mycoplasma was the dominant genus in the healthy group, Candidatus Xenohaliotis californiensis was dominant in the WS group. However, Candidatus Xenohaliotis californiensis was present in two healthy specimens, and thus the Mycoplasma/Candidatus Xenohaliotis californiensis ratio appears to be more determinant in specimens affected with WS. Further research to elucidate the role of digestive gland microbiota ecology in WS pathogenesis is required.
Project description:Vibrio species represent one of the most diverse genera of marine bacteria known for their ubiquitous presence in natural aquatic systems. Several members of this genus including Vibrio harveyi are receiving increasing attention lately because they are becoming a source of health problems, especially for some marine organisms widely used in sea food industry. To learn about adaptation changes triggered by V. harveyi during its long-term persistence at elevated temperatures, we studied adaptation of this marine bacterium in sea water microcosms at 30 oC that closely mimicks the upper limits of sea surface temperatures recorded around the globe. Overall design: Total RNA was isolated from Vibrio harveyi ATCC 14126 cultures by triplicate. To carry out this analysis, total RNA was isolated from the initial inoculate grown in marine broth at 26ºC (control) and from V. harveyi cells after their permanence in sterile seawater at 30ºC for 12 hours, 3 and 6 days (Stress). RNA samples were submitted for sequencing. Read were aligned to Vibrio harveyi ATCC 43516 genome.
Project description:Two new species of the genus Rissoella Gray, 1847 are described from Venezuela, one from the National Park Morrocoy, Rissoella morrocoyensissp. n. and the other from the Wildlife Refuge Isla de Aves, Rissoella venezolanicolasp. n.Rissoella morrocoyensissp. n. has a deep umbilicus (partly closed), preumbilical cord, black head, hypobranchial gland marked by a pale yellow boomerang-shaped ribbon and it lives on the leaves of the seagrass Thalassia testudinum Banks & König, 1805. Rissoella venezolanicolasp. n. has an angled preumbilical cord which extends to the columella delimiting a trapezoid, a hypobranchial gland marked by a yellow quaver-shaped ribbon and protoconch with fuchsia highlights. It lives on the brown alga Dictyota spp. The records of Rissoella in the Caribbean are revised and illustrations, a comparative table and a key to the Caribbean species known for the genus are provided.
Project description:Functional hypoxia is a stress condition caused by the abalone itself as a result of increased muscle activity, which generally necessitates the employment of anaerobic metabolism if the activity is sustained for prolonged periods. With that being said, abalone are highly reliant on anaerobic metabolism to provide partial compensation for energy production during oxygen-deprived episodes. However, current knowledge on the holistic metabolic response for energy metabolism during functional hypoxia, and the contribution of different metabolic pathways and various abalone tissues towards the overall accumulation of anaerobic end-products in abalone are scarce. Metabolomics analysis of adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue samples indicated that South African abalone (Haliotis midae) subjected to functional hypoxia utilises predominantly anaerobic metabolism, and depends on all of the main metabolite classes (proteins, carbohydrates and lipids) for energy supply. Functional hypoxia caused increased levels of anaerobic end-products: lactate, alanopine, tauropine, succinate and alanine. Also, elevation in arginine levels was detected, confirming that abalone use phosphoarginine to generate energy during functional hypoxia. Different tissues showed varied metabolic responses to hypoxia, with functional hypoxia showing excessive changes in the adductor muscle and gills. From this metabolomics investigation, it becomes evident that abalone are metabolically able to produce sufficient amounts of energy when functional hypoxia is experienced. Also, tissue interplay enables the adjustment of H. midae energy requirements as their metabolism shifts from aerobic to anaerobic respiration during functional hypoxia.This article has an associated First Person interview with the first author of the paper.
Project description:Vibrio bacteria, and particularly members of the Harveyi clade, are the causative agents of vibriosis. This disease is responsible for mass mortality events and important economic losses on aquaculture farms. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. 16S rRNA gene sequencing is generally considered to be the gold standard for bacterial identification but the cost and long processing time make it difficult to apply for routine identification. In contrast, MALDI-TOF MS offers rapid diagnosis and is commonly used in veterinary laboratories today. The major limiting factor for using this technique is the low environmental bacterial diversity in the commonly available databases. Here, we demonstrate that the sole use of the commercially available Bruker BioTyper database is not fully adequate for identifying Vibrio bacteria isolated from aquaculture farms. We therefore developed a new in-house database named Luvibase, composed of 23 reference MALDI-TOF mass spectra profiles obtained from Vibrio collection strains, mostly belonging to the Harveyi clade. The comparison of the accuracy of MALDI-TOF MS profiling and 16S rRNA gene sequencing revealed a lack of resolution for 16S rRNA gene sequencing. In contrast, MALDI-TOF MS profiling proved to be a more reliable tool for resolving species-level variations within the Harveyi clade. Finally, combining the Luvibase with the Bruker ver.188.8.131.52 database, led to successful identification of 47 Vibrio isolates obtained from moribund abalone, seabass and oysters. Thus, the use of Luvibase allow for increased confidence in identifying Vibrio species belonging to the Harveyi clade.