Freshwater Strains Research Articles

Towards deciphering dynamic changes and evolutionary mechanisms involved in the adaptation to low salinities in Ectocarpus (brown algae).

Colonizations of freshwater by marine species are rare events, and little information is known about the underlying mechanisms. Brown algae are an independent lineage of photosynthetic and multicellular organisms from which few species inhabit freshwater. As a marine alga that is also found in freshwater, Ectocarpus is of particular interest for studying the transition between these habitats. To gain insights into mechanisms of the transition, we examined salinity tolerance and adaptations to low salinities in a freshwater strain of Ectocarpus on physiological and molecular levels. We show that this isolate belongs to a widely distributed and highly stress-resistant clade, and differed from the genome-sequenced marine strain in its tolerance of low salinities. It also exhibited profound, but reversible, morphological, physiological, and transcriptomic changes when transferred to seawater. Although gene expression profiles were similar in both strains under identical conditions, metabolite and ion profiles differed strongly, the freshwater strain exhibiting e.g. higher cellular contents of amino acids and nitrate, higher contents of n-3 fatty acids, and lower intracellular mannitol and sodium concentrations. Moreover, several stress markers were noted in the freshwater isolate in seawater. This finding suggests that, while high stress tolerance and plasticity may be prerequisites for the colonization of freshwater, genomic alterations have occurred that produced permanent changes in the metabolite profiles to stabilize the transition.

Evaluation of flocculants for the recovery of freshwater microalgae

The use flocculants on the recovery of freshwater microalgae is studied. Flocculants tested include metal salts, chitosan, and polyelectrolytes used in wastewater treatment processes. Influence of flocculant, but also the doses and biomass concentrations affecting biomass recovery as well as the concentration factor has been evaluated. Results showed that the use of metal salts or chitosan was not efficient, whereas polyelectrolytes allow the efficient recovery of biomass, at doses of 2–25mg per gram of microalgae biomass. The required doses depend on the particular polyelectrolyte and the freshwater strain present; but cationic polyelectrolytes are generally recommended. The use of polyelectrolytes does not adversely affect water reuse in the production process. The concentration factors obtained are higher than 35 in most cases. Such high concentration factors allow a reduction in the equipment size necessary for biomass dewatering, thus improving the viability of using these microorganisms in biofuel or wastewater processes.

The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.

Bringing Planctomycetes into pure culture

Planctomycetes have been known since the description of Planctomyces bekefii by Gimesi at the beginning of the twentieth century (1924), although the first axenic cultures were only obtained in the 1970s. Since then, 11 genera with 14 species have been validly named and five candidatus genera belonging to the anaerobic ammonium oxidation, anammox bacteria have also been discovered. However, Planctomycetes diversity is much broader than these numbers indicate, as shown by environmental molecular studies. In recent years, the authors have attempted to isolate and cultivate additional strains of Planctomycetes. This paper provides a summary of the isolation work that was carried out to obtain in pure culture Planctomycetes from several environmental sources. The following strains of planctomycetes have been successfully isolated: two freshwater strains from the sediments of an aquarium, which were described as a new genus and species, Aquisphaera giovannonii; several Rhodopirellula strains from the sediments of a water treatment recycling tank of a marine fish farm; and more than 140 planctomycetes from the biofilm community of macroalgae. This collection comprises several novel taxa that are being characterized and described. Improvements in the isolation methodology were made in order to optimize and enlarge the number of Planctomycetes isolated from the macroalgae. The existence of an intimate and an important relationship between planctomycetes and macroalgae reported before by molecular studies is therefore supported by culture-dependent methods.

Mining Genomes of Marine Cyanobacteria for Elements of Zinc Homeostasis

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co2+ and Cd2+. Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs).

Cyanobacteria of the genus prochlorothrix.

Green cyanobacteria differ from the blue–green cyanobacteria by the possession of a chlorophyll-containing light-harvesting antenna. Three genera of the green cyanobacteria namely Acaryochloris, Prochlorococcus, and Prochloron are unicellular and inhabit marine environments. Prochlorococcus marinus attracts most attention due to its prominent role in marine primary productivity. The fourth genus Prochlorothrix is represented by the filamentous freshwater strains. Unlike the other green cyanobacteria, Prochlorothrix strains are remarkably rare: to date, living isolates have been limited to two European locations. Taking into account fluctuating blooms, morphological resemblance to Planktothrix and Pseudanabaena, and unsuccessful attempts to obtain enrichments of Prochlorothrix, the most successful strategy to search for this cyanobacterium involves PCR with environmental DNA and Prochlorothrix-specific primers. This approach has revealed a broader distribution of Prochlorothrix. Marker genes have been found in at least two additional locations. Despite of the growing evidence for naturally occurring Prochlorothrix, there are only a few cultured strains with one of them (PCC 9006) being claimed to be axenic. In multixenic cultures, Prochlorothrix is accompanied by heterotrophic bacteria indicating a consortium-type association. The genus Prochlorothrix includes two species: P. hollandica and P. scandica based on distinctions in genomic DNA, cell size, temperature optimum, and fatty acid composition of membrane lipids. In this short review the properties of cyanobacteria of the genus Prochlorothrix are described. In addition, the evolutionary scenario for green cyanobacteria is suggested taking into account their possible role in the origin of simple chloroplast.

Isolation of wild microalgae from natural water bodies for high hydrogen producing strains

Water samples from various marine, freshwater and terrestrial environments in China have been collected and screened in our laboratory for wild clones of microalgae since 2008. Hundreds of microalgal clones were purified through plate streaking, of which 52 strains have been screened for their ability to produce H2 under anaerobic conditions. Twenty eight strains including 13 freshwater green algae, 12 marine green algae and 3 cyanobacteria were found to evolve H2 under sulfur or nitrogen deprivation. Parietochloris incisa, Chlorella protothecoides, Chlorella capsulata, Nannochloropsis sp., Tetraselmis helgolandica, Pyramimonas sp., Chlorella autotrophica, Dunaliella apiculata, Tetraselmis striata and Tetraselmis tetrathele were first reported as new H2 producing species. Among those H2 producing strains, freshwater C. protothecoides showed the highest H2 producing capacity (2.93 H2 ml/l/h) and accumulated up to 123.6 ml H2 per liter culture under sulfur deprivation. Marine C. autotrophica and T. striata were two notable green marine species that produced H2 phototrophically, with 3.73 ml/l and 20.11 ml/l of H2 yield respectively. Our results also demonstrated that both the likelihood of a strain producing H2, and the average H2 output, in freshwater strains were much higher than that in marine strains. In addition, the time for inducing H2 production in freshwater strains was short compared to the marine ones. All three Arthrospira strains tested were able to generate H2 under sulfur (S-) deprivation though the cultures would quickly lose their vitality in S-deprived medium. Interestingly, Arthrospira produced H2 under nitrogen deprivation, and the H2 yield was even higher than under S-depletion. However, neither tested chrysophyte nor diatom produced any H2 under anaerobiosis caused by S-deprivation.

Membrane topology of the cyanobacterial bicarbonate transporter, SbtA, and identification of potential regulatory loops

The transporter SbtA is a high affinity Na+-dependent HCO3- uptake system present in a majority of cyanobacterial clades. It functions in conjunction with CO2 uptake systems and other HCO3- uptake systems to allow cyanobacteria to accumulate high levels of HCO3- used to support efficient photosynthetic CO2 fixation via the CO2 concentrating mechanism in these species. The phoA/lacZ fusion reporter method was used to determine the membrane topology of the cyanobacterial bicarbonate transporter, SbtA (predicted size of ∼39.7 kD), cloned from the freshwater strain, Synechocystis PCC6803. The structure conforms to a model featuring 10 transmembrane helices (TMHs), with a distinct 5+5 duplicated structure. Both the N- and C-terminus are outside the cell and the second half of the protein is inverted relative to the first. The first putative helix appears to lack sufficient topogenic signals for its correct orientation in the membrane and instead relies on the presence of later helices. The cytoplasmic loop between helices 5 and 6 is a likely location for regulatory mechanisms that could govern activation of the transporter, and the cytoplasmic loop between helices 9 and 10 also contains some conserved putative regulatory residues.

A freshwater cyanophage whose genome indicates close relationships to photosynthetic marine cyanomyophages.

Bacteriophage S-CRM01 has been isolated from a freshwater strain of Synechococcus and shown to be present in the upper Klamath River valley in northern California and Oregon. The genome of this lytic T4-like phage has a 178,563 bp circular genetic map with 297 predicted protein-coding genes and 33 tRNA genes that represent all 20-amino-acid specificities. Analyses based on gene sequence and gene content indicate a close phylogenetic relationship to the 'photosynthetic' marine cyanomyophages infecting Synechococcus and Prochlorococcus. Such relatedness suggests that freshwater and marine phages can draw on a common gene pool. The genome can be considered as being comprised of three regions. Region 1 is populated predominantly with structural genes, recognized as such by homology to other T4-like phages and by identification in a proteomic analysis of purified virions. Region 2 contains most of the genes with roles in replication, recombination, nucleotide metabolism and regulation of gene expression, as well as 5 of the 6 signature genes of the photosynthetic cyanomyophages (hli03, hsp20, mazG, phoH and psbA; cobS is present in Region 3). Much of Regions 1 and 2 are syntenic with marine cyanomyophage genomes, except that a segment encompassing Region 2 is inverted. Region 3 contains a high proportion (85%) of genes that are unique to S-CRM01, as well as most of the tRNA genes. Regions 1 and 2 contain many predicted late promoters, with a combination of CTAAATA and ATAAATA core sequences. Two predicted genes that are unusual in phage genomes are homologues of cellular spoT and nusG.

Analysis of cultivable aerobic bacteria isolated from bottom sediments in the Wijdefjorden region, Spitsbergen

Analysis of cultivable aerobic bacteria isolated from bottom sediments in the Wijdefjorden region, SpitsbergenThe paper presents the first physicochemical and microbiological studies conducted in the northern area of Svalbard (Spitsbergen). Ten sediment samples were collected from the bottom of the longest fjord in the region, Wijdefjorden. Bottom sediments from ten lakes lo- cated along the shores of Wijdefjorden and Woodfjorden were also sampled. Organic matter content (LOI), water content, temperature, pH, and salinity of the sediments were determined. The quantity of aerobic bacteria cultured on various growth media at 4°C, 14°C, and 37°C ranged from 102to 106cfu/g of wet sediment mass, depending on the type of sampling station (fjord or lake). The number of bacteria did not correlate with organic matter content. Out of the 37 bacterial strains isolated from Wijdefjorden, 48% and 70% revealed ureolytic and proteolytic activity, respectively. The proportion of freshwater strains with ureolytic and proteolytic activitywas 32% and 55%, respectively. Antibiotic resistance testing indicated that bacterial strains from the bottom sediments of the lakes were resistant to 8 antibiotics (out of the 18 investigated). Possible sources of this resistance are discussed. Using 16S DNA analysis, bacterial isolates from the lakes were identified asPseudomonassp., whereas frequently occurring strains in bottom sediment of the fjord werePseudoalteromonassp.

ChemInform Abstract: The Unique Mechanistic Transformations Involved in the Biosynthesis of Modular Natural Products from Marine Cyanobacteria

AbstractReview: 107 refs.

Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application

We describe a methodology to investigate the potential of given microalgae species for biodiesel production by characterizing their productivity in terms of both biomass and lipids. A multi-step approach was used: determination of biological needs for macronutrients (nitrate, phosphate and sulphate), determination of maximum biomass productivity (the "light-limited" regime), scaling-up of biomass production in photobioreactors, including a theoretical framework to predict corresponding productivities, and investigation of how nitrate starvation protocol affects cell biochemical composition and triggers triacylglycerol (TAG) accumulation. The methodology was applied to two freshwater strains, Chlorella vulgaris and Neochloris oleoabundans, and one seawater diatom strain, Cylindrotheca closterium. The highest total lipid content was achieved with N. oleoabundans (25-37% of DW), while the highest TAG content was found in C. vulgaris (11-14% of DW). These two species showed similar TAG productivities.

Expression of hcp in freshwater Synechococcus spp., a gene encoding a hyperconserved protein in picocyanobacteria

Marine picoplankton of the genus Synechococcus and Prochlorococcus spp. are widely studied members of the picocyanobacterial clade, composed of unicellular cyanobacteria that dominate pelagic regions of the ocean. Less studied are the related freshwater Synechococcus spp. that similarly dominate the euphotic zone of oligotrophic lakes. Previous work has shown that marine picocyanobacteria harbor a small gene, hcp, that encodes a 62 amino acid protein 100% conserved among all strains examined. The gene is restricted exclusively to the picocyanobacterial lineage. The current study reveals that hcp is also 100% conserved in four freshwater Synechococcus spp. strains isolated from the Laurentian Great Lakes, and that the gene constitutively expressed with genes encoding a ribosomal protein and two tRNA genes. The synteny of the hcp region is also conserved between the marine and freshwater strains. Last, the hcp gene and the organization of the surrounding genetic region has been retained in the reduced genome of a picocyanobacterial endosymbiont of the amoeba Paulinella sp.

Molybdenum—nitrogen co‐limitation in freshwater and coastal heterocystous cyanobacteria

Molybdenum (Mo) is essential for the biological assimilation of inorganic nitrogen (N). We compared Mo requirements for N2‐fixation in two species of filamentous heterocystous cyanobacteria (HC) to test the hypothesis that coastal HC require higher Mo concentrations than freshwater HC. This expectation follows from the fact that Mo is more concentrated in seawater (~ 105 nmol L−1) than in most freshwaters (< 20 nmol L−1). Contrary to this hypothesis, we found that both strains maintained N2‐fixation for 30 d at 10 nmol L−1. Mo concentrations < 1 nmol L−1 induced N‐limitation in both species, as indicated by increased C:N ratios and decreased nitrogenase expression and activity. This response took time to induce, likely due to high‐affinity molybdate uptake by both species. Measurable N2‐fixation persisted in the coastal strain (Nostoc sp. CCMP 2511) for at most 12 d; 3 d were required for chlorophyll a concentrations to fall below those of Mo‐replete cultures. An additional 7 d and 11 d, respectively, were required for N2‐fixation rates and chlorophyll levels to decline in Mo‐limited freshwater cultures (Nostoc sp. PCC 7120). When Mo was high (> 1 µmol L−1), the freshwater strain exhibited considerable Mo storage (> 100 µmol mol−1 Mo: C) whereas cellular Mo remained < 10 µmol mol−1 Mo:C in the coastal strain. The high Mo content and extended time required for N2‐fixation to decrease in the freshwater strain could be due to expression of the gene mop, which encodes a putative molybdate‐storage protein. This study suggests the importance of Mo storage in freshwater HC.

Culturability and Survival of Marine, Freshwater and Clinical Pseudomonas aeruginosa

Genetic typing of Pseudomonas aeruginosa isolated from the open ocean has revealed that marine strains form unique clusters. To clarify whether this genetic variation reflects differences in pattern of culturability and survival, a marine strain was compared with a freshwater strain and a clinical strain in microcosms with different levels of NaCl (0 to 7% [w/v]), pH (4.0 to 9.0) and temperature (-20, 0, 4, 25 and 37°C) in both artificial seawater (ASW) and distilled water (DW). The viable but non-culturable (VBNC) state of P. aeruginosa was also monitored. The marine strain 1200 grew better at high NaCl and pH, whereas the freshwater strain 1030 did better at 0 to 3% NaCl and a pH of less than 7.0. The clinical strain 1564 grew best at neutral pH and 0% NaCl. No significant differences were observed among the strains in culturability at different temperatures. Like other bacteria, P. aeruginosa enters a VBNC state under stressful conditions. The marine P. aeruginosa isolate exhibits a unique pattern of culturability and survival which demonstrates a physiological adaptation to the ocean environment.

The Estela Sousa e Silva Algal Culture Collection: a resource of biological and toxicological interest

The Estela Sousa e Silva Algal Culture Collection (ESSACC) is the legacy of nearly 40 years of the National Institute of Health Dr. Ricardo Jorge (INSA) scientific and public activity in marine and freshwater phytoplankton biology and toxicology. The living isolates maintained in the ESSACC are mostly represented by marine dinoflagellates and freshwater cyanobacteria strains isolated from bloom occurrences in Portugal. More than 170 isolates comprising the most frequently found species have been obtained from environmental samples and are successfully cultured at INSA facilities. Moreover, new isolates are continuously being added to the collection, following new detection of natural blooms. Although not intended to represent the entire range of taxonomical different species occurring in Portuguese environments, the ESSACC includes a wide array of geographical, morphological, physiological, and ecological relevant isolates. So far, laboratory up-scaled culturing has been put in place for several purposes, including the production of secondary metabolites, purification of toxins, detection of toxin producing strains by molecular methods, screening for cytotoxic and genotoxic effects of purified compounds, testing for antialgal activity of organic compounds, and evaluating the combining effects of environmental factors on biomass and toxin production. We are disseminating information about this collection since it is an important wide source of readily available and easy to manipulate biological material for research purposes within the scientific community.

Investigation of antimicrobial and protease-inhibitory activity from cultured cyanobacteria

A culture collection of cyanobacteria has been established at the University of Illinois at Chicago. This collection includes marine, terrestrial, and freshwater strains and contains representatives of the five orders of cyanobacteria: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, and Stigonematales. In this study, extracts from a subset of 61 strains, 16 marine and 45 freshwater/terrestrial, were evaluated against three current protease targets, i.e. 20S proteasome and two SARS viral proteases, two important bacterial targets, i.e. Mycobacterium tuberculosis and Bacillus anthracis, and in the Artemia salina toxicity assay. In total, extracts of 12 strains possessed significant levels of activity in one or more targets. The overwhelming majority of active extracts (11 of 12) were from either freshwater or terrestrial forms of cyanobacteria, with the greater part of these (9 of 12) being heterocyst-forming strains. These results further support the use of cultured cyanobacteria as a source of biologically active natural products.

Cloning and tissue-specific expression of a .DELTA.-COP homologue in a freshwater and a brackish water-adapted strain of rainbow trout (Oncorhynchus mykiss)

In eukaryotic cells, intracellular transport is mediated by coated vesicular carriers. Coat proteins I (COPI) vesicles are involved in the retrograde transport from Golgi apparatus to the endoplasmic reticulum. The COPI complex is composed of ADP-ribosylation factor 1 and coatomer comprising seven subunits, termed alpha-zeta. We isolated and characterised a cDNA sequence from rainbow trout homolog to delta-subunit of COPI complex (delta-COP). Trout delta-COP gene encodes a protein of 509 aa including a characteristic Mu homology domain. Searches at the Ensemble Genome browser identified three additional teleostean delta-COP-like sequences from pufferfish, rice fish, and stickleback. Sequence identity of piscine delta-COP protein sequences is greater than 84%. Moreover, a phylogenetic analysis indicates that delta-COP protein sequences are strongly conserved among vertebrate species. delta-COP homologue is ubiquitously expressed in trout tissues. Quantitative Real-Time RT-PCR revealed that delta-COP is differentially expressed in liver and gill tissue of two rainbow trout strains, the freshwater strain STEELHEAD and the brackish water-adapted strain BORN.

Nitrogen and oxygen isotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria

We report the first measurements of coupled nitrogen (N) and oxygen (O) isotope fractionation of nitrate by laboratory cultures of denitrifying bacteria. Two seawater strains (Pseudomonas stutzeri, Ochrobactrum sp.) and three freshwater strains (Paracoccus denitrificans, Pseudomonas chlororaphis, Rhodobacter sphaeroides) were examined. Among four strains of facultative anaerobic denitrifiers, N and O isotope effects were variable, ranging from 5‰ to 25‰, with evidence for a drop in the isotope effects as nitrate concentrations approached the halfsaturation constant for nitrate transport. O isotope effects were similar to their corresponding N isotope effect, such that the progressive increase in nitrate δ18O, when plotted against that in δ15N (where δ18Osample = [(δ18O: δ16Osample/(δ18O:δ16O)reference ‐ 1] × 1000, and δ15Nsample × [(15N:14N)sample/(δ15N:δ14N)reference ‐ 1] × 1000), yielded slopes of 0.86 to 1.02, with a mean value of 0.96. R. sphaeroides, a photo‐heterotroph that possesses only a periplasmic (nonrespiring) dissimilatory nitrate reductase, showed less variability in nitrate N isotope effects, between 13‰ and 20‰, with a modal value of ~15‰. In contrast to the respiratory denitrifiers, R. sphaeroides consistently showed a distinct ratio of δ18O to δ15N change of ~0.62. We hypothesize that heavy N and O isotope discrimination during respiratory denitrification occurs during the intracellular reduction of nitrate by the respiratory nitrate reductase, and the observed magnitude of fractionation is likely regulated by the ratio of cellular nitrate efflux relative to uptake. The data for R. sphaeroides are consistent with isotope discrimination directly reflecting the N and O isotope effects of the periplasmic nitrate reductase NAP, without modification by nitrate uptake and efflux.

Differences in Virulence of Marine and Freshwater Isolates of Viral Hemorrhagic Septicemia Virus In Vivo Correlate with In Vitro Ability To Infect Gill Epithelial Cells and Macrophages of Rainbow Trout ( Oncorhynchus mykiss )

Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.