Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in eastern oyster (Crassostrea virginica) hemocytes

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinas suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues

Osmotic Tolerance And Volume Regulation In In Vitro Cultures Of The Oyster Pathogen Perkinsus Marinus

Growth rate. cell size, osmotic tolerance, and volume regulation were examined in cells of Perkinsus marinus cultured in media of osmolalities ranging from 168 to 737 mOsm (6.5-27.0 ppt). Cells cultured at the low osmolalities of 168 and 256 mOsm (6.5 and 9.7 ppt) began log phase growth 4 days postsubculture, whereas cells cultured at the higher osmolalities 341, 433, and 737 mOsm (12.7. 16.0, and 27.0 ppt) began log phase growth 2 days postsubculture. During log phase growth, cells from the higher osmolalities 341, 433, and 737 mOsm had shorter doubling times than cells from the lower osmolalities 168 and 256 mOsm. During both log and stationary phase growth, the mean cell diameter of cells cultured at 168 mOsm was significantly greater than cells cultured at 341 and 737 mOsm; the mean diameters of cells cultured at 341 and 737 mOsm did not differ significantly from each other. P. marinus cells cultured in various osmolalities were exposed to artificial seawater treatments of 56-672 mOsm (2.5-24.7 ppt). After the hypoosmotic treatment of 56 mOsm, cells that had been cultured in medium of low osmolality, 168 mOsm, showed only 41% mortality whereas the cells from the 341-, 433-, and 737-mOsm culture groups experienced 100% mortality. During the hyperosmotic shock, all of the groups exhibited mortalities of less than 10%. In P. marinus cells cultured in medium of 737 mOsm and then placed in a 50% dilution, cell diameter increased 13% which was a volume increase of 44.5%, but cells returned to baseline size (size before osmotic shock) within 5 minutes. P. marinus cells cultured at low osmolalities can withstand both hypo- and hyperosmotic stress and use volume-regulatory mechanisms during hypoosmotic stress. Results suggest that transferring infected oysters to low salinity will result in strains of P. marinus acclimated to low salinity that will be able to withstand periodic events of extremely low salinity

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases

Alignment of the ALICE Inner Tracking System with cosmic-ray tracks

37 pages, 15 figures, revised version, accepted by JINSTALICE (A Large Ion Collider Experiment) is the LHC (Large Hadron Collider) experiment devoted to investigating the strongly interacting matter created in nucleus-nucleus collisions at the LHC energies. The ALICE ITS, Inner Tracking System, consists of six cylindrical layers of silicon detectors with three different technologies; in the outward direction: two layers of pixel detectors, two layers each of drift, and strip detectors. The number of parameters to be determined in the spatial alignment of the 2198 sensor modules of the ITS is about 13,000. The target alignment precision is well below 10 micron in some cases (pixels). The sources of alignment information include survey measurements, and the reconstructed tracks from cosmic rays and from proton-proton collisions. The main track-based alignment method uses the Millepede global approach. An iterative local method was developed and used as well. We present the results obtained for the ITS alignment using about 10^5 charged tracks from cosmic rays that have been collected during summer 2008, with the ALICE solenoidal magnet switched off.Peer reviewe

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray\u27s fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination, The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile bi-flagellated zoospores, was observed at a low frequency (\u3c 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol\u27s solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham\u27s F-12 with fetuin

Transcriptome of American Oysters, \u3cem\u3eCrassostrea virginica\u3c/em\u3e, in Response to Bacterial Challenge: Insights into Potential Mechanisms of Disease Resistance

The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance

Habitat Modification and Coastal Protection by Ecosystem-Engineering Reef-Building Bivalves

Reef-building bivalves like oysters and mussels are conspicuous ecosystemengineers in intertidal and subtidal coastal environments. By forming complex,three-dimensional structures on top of the sediment surface, epibenthic bivalvereefs exert strong bio-physical interactions, thereby influencing local hydro- and morphodynamics as well as surrounding habitats and associated species. The spatial impact of the ecosystem engineering effects of reef-building bivalves is much larger than the size of the reef. By influencing hydrodynamics oysters and mussels modify the sedimentary environment far beyond the boundaries of the reef, affecting morphological and ecological processes up to several hundreds of meters. Being key-stone species in many coastal environments, reef-building bivalves are increasingly recognized for their role in delivering important ecosystem services that serve human wellbeing. Here we focus on two services, namely the regulating service coastal protection (coastal erosion prevention, shoreline stabilization) and the supporting habitat for species service (enhancement of biodiversity and diversification of the landscape). Due to their wave dampening effects, reef-building bivalve reefs are increasingly used for shoreline protection and erosion control along eroding coastlines, as an alternative to artificial shoreline hardening. Thefacilitative interactions at long-distances of bivalve reefs provide biodiversity benefits and more specifically facilitate or protect other valuable habitats such as intertidal flats, sea grasses, saltmarshes and mangroves. Two case studies are used to demonstrate how bivalve reefs can be restored or constructed for shoreline protection and erosion control, thereby focusing on oyster reefs: (1) Oyster reefs for shoreline protection in coastal Alabama, USA, and (2) Oyster reefs as protection against tidal flat erosion, Oosterschelde, The Netherlands. It is argued that bivalve reefs should be promoted as nature-based solutions that provide biodiversity benefits and coastal protection and help in climate change mitigation and adaptation. In order to successfully restore these habitats practitioners should consider a general framework in which habitat requirements, environmental setting and long-distance interdependence between habitats are mutually considered