Analyses of Kings Creek Water and Watershed Runoff Samples for Bacteroidales using qPCR to Detect Human Fecal Contamination

The purpose of this work was to evaluate and analyze water samples collected from the Kings Creek watershed using a qPCR-based method to detect both total Bacteroidales and Bacteroidales reported to be associated with human fecal contamination. Quantitative real-time PCR assays were used to significantly reduce processing times and at the same time yield estimates of target concentrations. Initial efforts focused on evaluation of various Bacteroidales primer sets reported in the literature tested against human and animal fecal samples collected from the Kings Creek watershed. Most samples, both animal and human, were positive with the universal (i.e. general or total) Bacteroidales assay. Strong positive signals were found with human sewage using the humanspecific assay that was chosen for this study. Most animal scat samples were negative with respect to the human-specific Bacteroidales indicator. The few animal samples that were positive with the human-specific assay had very low signal intensity. Despite the generally pervasive drought conditions during this study, evidence of human contamination was detected at certain feeder stream locations, and was widespread after a significant rain event that occurred in late fall. Use of the human-specific Bacteroidales indicator holds promise as a tool to identify potential human, as opposed to animal, sources of contamination but will require a more comprehensive field monitoring and sample collection effort than could be managed in this preliminary study

Effect Of Homogenate From Different Oyster Species On Perkinsus Marinus Proliferation And Subtilisin Gene Transcription

The modulation of Perkinsus marinus proliferation and subtilisin gene transcription by host (oyster) tissue was examined. Perkinsus rnarinus cells were cultured for 4 weeks in media supplemented with extract from either one of four different Crassostrea virginica stocks or with extract from one of two other Crassostrea species, C. ariakensis and C. gigas. After 4 weeks in culture, we determined cell counts and relative subtilisin gene transcription levels using quantitative real-time polymerise chain reaction (qRTPCR). Cell proliferation and subtilisin gene transcription were significantly lower when P. marinus\u27 cells were grown in the presence of homogenate from any of the three oyster species than in unsupplemented media. Perkinstrs marinas subtilisin gene transcription was also significantly lower in cells cultured in media supplemented with homogenate from either C. ariakensis or C. gigas, than in media containing extract from the native oyster host, C. virginica. Gene transcription levels among cells grown in media supplemented with homogenate from the different stocks of C. virginica were not significantly different from one another

A Deterministic Model for Understanding Nonlinear Viral Dynamics in Oysters

Contamination of oysters with a variety of viruses is one key pathway to trigger outbreaks of massive oyster mortality as well as human illnesses, including gastroenteritis and hepatitis. Much effort has gone into examining the fate of viruses in contaminated oysters, yet the current state of knowledge of nonlinear virus-oyster interactions is not comprehensive because most studies have focused on a limited number of processes under a narrow range of experimental conditions. A framework is needed for describing the complex nonlinear virus-oyster interactions. Here, we introduce a mathematical model that includes key processes for viral dynamics in oysters, such as oyster filtration, viral replication, the antiviral immune response, apoptosis, autophagy, and selective accumulation. We evaluate the model performance for two groups of viruses, those that replicate in oysters (e.g., ostreid herpesvirus) and those that do not (e.g., norovirus), and show that this model simulates well the viral dynamics in oysters for both groups. The model analytically explains experimental findings and predicts how changes in different physiological processes and environmental conditions nonlinearly affect in-host viral dynamics, for example, that oysters at higher temperatures may be more resistant to infection by ostreid herpesvirus. It also provides new insight into food treatment for controlling outbreaks, for example, that depuration for reducing norovirus levels is more effective in environments where oyster filtration rates are higher. This study provides the foundation of a modeling framework to guide future experiments and numerical modeling for better prediction and management of outbreaks

Advanced Perkinsus Marinus Infections In Crassostrea Ariakensis Maintained Under Laboratory Conditions

The Suminoe oyster, Crassostrea ariakensis, has been under investigation since the early 1990s for potential use in restoring the commercial harvest or for aquaculture of oysters in the Chesapeake Bay, USA. Initial studies focusing on C. ariakensis documented a significant level of tolerance to the protozoan parasite Perkinsus marinus, a pathogen found in almost all reaches of the Bay and widely acknowledged as one of the main reasons for the decline in the eastern oyster, Crassostrea virginica, harvest since the late 1980s. Crassostrea ariakensis was demonstrated to acquire P. marinus. however infection intensities, as measured using Ray\u27s thioglycollate medium assay indices, generally were found to be light. As part of a series of experiments to study potential impacts on the Chesapeake Bay region of pathogens found in C. ariakensis in Asia, a challenge experiment was conducted to study the pathogenicity of Perkinsus olseni to C. ariakensis. During this study, we observed the acquisition of moderate and heavy infection intensities of P. marinus in triploid C. ariakensis oysters being maintained in the laboratory. Results suggest that there may be some risk of mortality from P. marinus if C. ariakensis is held under stressful conditions at least in hatchery or laboratory settings

Wild and aquaculture populations of the eastern oyster compared using microsatellites

Five new microsatellite markers were developed for the eastern oyster (Crassostrea virginica), and allelic variability was compared between a wild Chesapeake Bay population (James River) and a hatchery strain (DEBY (TM)). All loci amplified readily and demonstrated allelic variability with the number of alleles ranging from 16 to 36 in the wild population and from 11 to 19 in the DEBY (TM) strain. Average observed and expected heterozygosities were estimated at 0.66 and 0.80 in the hatchery sample. The corresponding estimates were 0.91 and 0.75 in the wild sample. Results indicated lower genetic variability in the DEBY (TM) strain and significant genetic differentiation between the wild population and hatchery strain. These microsatellite loci will prove valuable for future population genetic studies and in tracking of hatchery strains used in restoration

Discrimination Of Nine Crassostrea Oyster Species Based Upon Restriction Fragment-Length Polymorphism Analysis Of Nuclear And Mitochondrial Dna Markers

A molecular genetic identification key for nine species of Crassostrea oysters was developed based on restriction fragment-length polymorphism (RFLP) analyses of nuclear and mitochondrial DNA markers. Seven of nine species were unambiguosly differentiated based on digestion of the ITS-1 nuclear marker with Hae III an Hinf I. Individual species exhibited one or two RFLP patterns for each restriction endonuclease, and only C. madrasensis and C. iredalei were indistinguishable electrophoretically. All nine species were unambiguosly differentiated based on digestion of the COI mitochondrial marker with Dde I and Hae III. Species exhibited one or two RFLP patterns for each restriction endonuclease, and species pairs unresolved by the first restriction enzyme were completely resolved with the second. The resulting key distinguishes among many Indo-Pacific Crassostrea oysters that overlap across some or all on their ranges, and establish as an expandable framework for future additions of other species to the key

Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea)

Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuca (Para state) to Santos (Sao Paulo state), and C. rhizophorae from Fortim (Ceara state) to Florianopolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Braganca, Para state). An unidentified Crassostrea species was found only on Canela Island, Braganca. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship

Actin Gene Family Evolution and the Phylogeny of Coleoid Cephalopods (Mollusca: Cephalopoda)

Phylogenetic analysis conducted on a 784-bp fragment of 82 actin gene sequences of 44 coleoid cephalopod taxa, along with results obtained from genomic Southern blot analysis, confirmed the presence of at least three distinct actin loci in coleoids. Actin isoforms were characteri zed through phylogenetic analysis of representative cephalopod sequences from each of the three isoforms, along with translated actin cDNA sequences from a diverse array of metazoan taxa downloaded from GenBank. One of the three isoforms found in cephalopods was closely related to actin sequences expressed in the muscular tissues of other molluscs. A second isoform was most similar to cytoplasmic-specific actin amino acid sequences. The muscle type actins of molluscs were found to be distinct from those of arthropods, suggesting at least two independent derivations of muscle actins in the protostome lineage, although statistical support for this conclusion was lacking. Parsimony and maximum-likelihood analyses of two of the isoforms from which \u3e30 orthologous coleoid sequences had been obtained (one of the cytoplasmic actins and the muscle actin) supported the monophyly of several higher-level coleoid taxa. These included the superorders Octopodiformes and Decapodiformes, the order Octopoda, the octopod suborder Incirrata, and the teuthoid suborder Myopsida. The monophyly of several taxonomic groups within the Decapodiformes was not supported, including the orders Teuthoidea and Sepioidea and the teuthoid suborder Oegopsida. Parametric bootstrap analysis conducted on the simulated cytoplasmic actin data set provided statistical support to reject the monophyly of the Sepioidea. Although parametric bootstrap analysis of the muscle actin isoform did not reject sepioid monophyly at the 5% level, the results (rejection at P = 0.068) were certainly suggestive of sepioid nonmonophyly