GENETIC POPULATION STRUCTURE OF PSEUDO-NITZSCHIA PUNGENS (BACILLARIOPHYCEAE) FROM THE PACIFIC NORTHWEST AND THE NORTH SEA(1).

Several species of the diatom Pseudo-nitzschia produce the neurotoxin domoic acid (DA). Consumption of fish and shellfish that have accumulated this potent excitotoxin has resulted in severe illness and even death in humans, marine mammals, and seabirds. Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle is a cosmopolitan diatom commonly occurring in the waters of the Pacific Northwest (PNW) and the eastern North Atlantic, including the North Sea. However, genetic and physiological relationships among populations throughout this large geographic distribution have not been assessed. Population genetic parameters (e.g., Hardy-Weinberg equilibrium, linkage equilibrium, FST ) calculated for P. pungens collected from the Juan de Fuca eddy region in the PNW indicated the presence of two distinct groups that were more divergent from each other than either was from a P. pungens sample from the North Sea. Geographic heterogeneity was also detected within each of the two PNW groups. These results suggested that the populations of P. pungens recently mixed in the Juan de Fuca eddy region (a seasonally retentive feature off the coasts of Washington State, USA, and Vancouver Island, Canada) but did not exchange genetic material by sexual reproduction. Alternatively, these two groups may be cryptic (morphologically identical, but reproductively isolated) species. Identifying cryptic diversity in Pseudo-nitzschia is important for bloom prediction and aiding the identification of molecular markers that can be used for rapid detection assay development.

Mesocosm experiments for evaluating the biological efficacy of ozone treatment of marine ballast water.

Ballast water is a major pathway for the transfer of non-indigenous species in aquatic environments. The objectives of this study were to determine the ability of ozone to reduce the numbers of a spectrum of marine organisms collected from Puget Sound, Washington in replicated mesocosm (280 l) experiments, and estimate the minimum ozone concentrations as measured by total residual oxidant (TRO) required to reduce organism densities. Ozone treatment was effective in removing bacteria, phytoplankton, and mesozooplankton with initial TRO concentrations of 2-5 mg l(-1) as Br(2). Persistence of TRO resulted in an extended period of toxicity and cumulative mortality. TRO decay allowed bacteria populations to multiply when TRO levels fell below 0.5-1.0 mg l(-1) as Br(2). Phytoplankton chlorophyll a concentrations were rapidly reduced by ozone treatment and did not increase in any treatments or controls because of lack of light. Overall mesozooplankton viability was rapidly reduced by 90-99% in treatment TRO levels above 1.85 mg l(-1) as Br(2). Our study outlines novel protocols that can be used for testing different potential ballast water treatment systems in replicated and controlled mesocosm experiments.

Ozonation of seawater from different locations: formation and decay of total residual oxidant--implications for ballast water treatment.

Ballast water is a likely cause for worldwide transfer of non-indigenous aquatic species because of the large volumes and frequency of possible inoculations. Ozone is one treatment option being considered for eliminating non-indigenous species in ballast water. When ozone is applied to seawater, secondary disinfectants are formed, commonly measured and expressed as total residual oxidant (TRO). The goal of this study was to determine those variables most likely to affect the rate of TRO increase during ozonation and the subsequent TRO decline that occurs over time. These parameters strongly influence the efficacy of ozone treatments aimed to eliminate organisms present in ballast water. Seawater was obtained from Puget Sound, Washington; Cape Fear, North Carolina; and San Francisco Bay. Results indicated that seawater characteristics, including the organic content and ammonia, affect the amount of ozone required to achieve a desired TRO level and rate of TRO decay, and therefore need to be considered in determining ozone requirements for ballast water treatment.

Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus.

The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination.

Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-alpha1, TNF-alpha2, IL-1beta1, IL-8, TGF-beta1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-beta1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae.

Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.

Pseudonocardia chloroethenivorans sp. nov., a chloroethene-degrading actinomycete.

A bacterial strain, SL-1T, capable of degrading trichloroethene was isolated from a laboratory enrichment in the Department of Civil and Environmental Engineering, University of Washington, USA. The material in the enrichments was derived from a soil sample from Seattle, WA, USA. Strain SL-1T was capable of using phenol as a source of carbon and energy. Chemotaxonomic, morphological, physiological and phylogenetic analyses showed that strain SL-1T is a member of the genus Pseudonocardia. The ability of strain SL-1T to utilize phenol and degrade trichloroethene, as well as other phenotypic properties and the results from a 16S rRNA phylogenetic analysis, led to the proposal of a novel species, Pseudonocardia chloroethenivorans sp. nov. The type strain is SL-1T (=ATCC BAA-742T=DSM 44698T). Trichloroethene and other chloroethenes are major pollutants at many environmental sites, and P. chloroethenivorans has biodegradation properties that should be of interest to environmental microbiologists and engineers.

High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex.

High hydrostatic pressure has been demonstrated to be a useful technique for treating food to reduce the number of pathogenic organisms and to extend shelf life. Most research in this area has focused on bacteria. However, a concern in the sashimi (raw fish) industry is that nematode worms such as Anisakis simplex occur naturally in cold-water marine fish. The objectives of this research were to perform a pilot study to determine the effect of high hydrostatic pressure on the viability of Anisakis simplex larvae, commonly found in king salmon and arrowtooth flounder, and to evaluate the effects of high hydrostatic pressure on the color and texture of the fish fillets. Pieces of fish (ca. 100 g per bag) containing 13 to 118 larvae were exposed to pressures of up to 80,000 lb/in2 (552 MPa) for up to 180 s. The times and pressures required to kill 100% of the larvae were as follows: 30 to 60 s at 60,000 lb/in2 (414 MPa), 90 to 180 s at 40,000 lb/in2 (276 MPa), and 180 s at 30,000 lb/in2 (207 MPa). For all salmon treatments that killed 100% of the larvae, a significant increase in the whiteness of the flesh was observed. Although high hydrostatic pressure was effective in killing A. simplex larvae in raw fish fillets, its significant effect on the color and overall appearance of the fillet may limit its application to the processing of fish for raw-fish markets.