Salmon Embryo Research Articles

Endocrine and developmental effects in Atlantic salmon ( Salmo salar) exposed to perfluorooctane sulfonic or perfluorooctane carboxylic acids

In this study, we have investigated the effect of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on endocrine signalling, growth and development in Atlantic salmon ( Salmo salar) embryos and larvae. Expression of genes related to the hypothalamic–pituitary–thyroid (HPT) axis, growth-hormone/insulin-like growth factor (GH/IGF) axis and the steroid hormone axis were used as indicators of endocrine disruption. We also studied bone development in larvae, both by observing skeletal structure formation and by investigating expression of genes involved in ossification process. Atlantic salmon embryos, kept in plastic tanks at 5–7 °C, were exposed to 100 μg/L PFOA or PFOS from egg stage for a period of 52 days, followed by one-week recovery period. Sampling was performed at day 21, 35, 49 and 56 representing age 549, 597, 679 and 721 dd (dd or day degrees = number of days × temperature in degree Celsius: °C). Note that day 56 or 721 dd is the end of the 1-week recovery period. Larvae were divided into designated head and body regions for the purpose of gene expression analysis, except for genes that regulate ossification that were analyzed in whole larvae. Expression of thyroid receptor α and β (TRα and TRβ), thyroid-stimulating hormone β (TSHβ), T 4 outer-ring deiodinase (T 4ORD), growth hormone (GH), insulin-like growth factor-I and II (IGF-I and II), insulin-like growth factor I receptor (IGF-IR), and estrogen receptor α and β (ERα and ERβ) were investigated using quantitative PCR. Both PFOS and PFOA exposure produced non-significant alterations in larvae weight (except after the recovery period when a decrease was observed), while larvae length was unaffected. PFOS and PFOA exposure produced body- and head region-specific alterations in expression of all the investigated gene transcripts. Expression of IGF-I and IGF-IR paralleled that of GH, indicating that perturbation of GH expression is a possible end point for disruption of the GH–IGF axis. We did not observe developmental changes related to angiogenesis, ossification and chondrogenesis after exposure to PFOS and PFOA. Transcriptional abnormalities may serve as indicators of chronic exposure, although the concrete mechanisms causing the observed effects remain ambiguous. The implications of these findings for the complete lifecycle, including other developmental and/or reproductive damage, are areas of future study.

A novel member of the family Hepeviridae from cutthroat trout ( Oncorhynchus clarkii)

Beginning in 1988, the Chinook salmon embryo (CHSE-214) cell line was used to isolate a novel virus from spawning adult trout in the state of California, USA. Termed the cutthroat trout ( Oncorhynchus clarkii) virus (CTV), the small, round virus was not associated with disease, but was subsequently found to be present in an increasing number of trout populations in the western USA, likely by a combination of improved surveillance activities and the shipment of infected eggs to new locations. Here, we report that the full length genome of the 1988 Heenan Lake isolate of CTV consisted of 7269 nucleotides of positive-sense, single-stranded RNA beginning with a 5′ untranslated region (UTR), followed by three open reading frames (ORFs), a 3′ UTR and ending in a polyA tail. The genome of CTV was similar in size and organization to that of Hepatitis E virus (HEV) with which it shared the highest nucleotide and amino acid sequence identities. Similar to the genomes of human, rodent or avian hepeviruses, ORF 1 encoded a large, non-structural polyprotein that included conserved methyltransferase, protease, helicase and polymerase domains, while ORF 2 encoded the structural capsid protein and ORF 3 the phosphoprotein. Together, our data indicated that CTV was clearly a member of the family Hepeviridae, although the level of amino acid sequence identity with the ORFs of mammalian or avian hepeviruses (13–27%) may be sufficiently low to warrant the creation of a novel genus. We also performed a phylogenetic analysis using a 262 nt region within ORF 1 for 63 isolates of CTV obtained from seven species of trout reared in various geographic locations in the western USA. While the sequences fell into two genetic clades, the overall nucleotide diversity was low (less than 8.4%) and many isolates differed by only 1–2 nucleotides, suggesting an epidemiological link. Finally, we showed that CTV was able to form persistently infected cultures of the CHSE-214 cell line that may have use in research on the biology or treatment of hepevirus infections of humans or other animals.

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L.

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface-related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S.phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S.phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.

Competitive interactions shape offspring performance in relation to seasonal timing of emergence in Atlantic salmon

1. Timing of birth/hatching may have strong effects on offspring fitness. Breeding time is generally considered to have evolved to match offspring arrival with optimal seasonal environmental conditions, though this is rarely tested experimentally and factors shaping the relations between timing of birth and reproductive success are often poorly understood. 2. By manipulating incubation temperature of Atlantic salmon embryos, and hence controlling for maternal and genetic effects, we obtained offspring emerging from nests prior to (accelerated), during and after (decelerated) normal emergence times, and accordingly experienced widely different seasonal environmental conditions at emergence (stream temperature range 4-16 °C). The accelerated group emerged at temperatures that are generally considered to be highly sub-optimal for growth and likely imposes strong constraints on feeding and activity, and during a peak in water discharge which is expected to negatively influence habitat availability. 3. In the wild, overall mortality during the period after emergence was 79%, and was significantly affected by both release density and emergence timing. Accelerated offspring, which emerged earliest and experienced the harshest environmental conditions, had both highest survival and largest final body size. The effect was particularly strong at the high density release site, where survival of accelerated offspring was significantly higher than both the normal and decelerated groups. 4. In more controlled semi-natural environments, all developmental groups were able to perform well, but accelerated offspring had a relatively better performance than the later emerging offspring when density was high. Furthermore, the relative performance of the different groups was not sensitive to water discharge regimes (temporally stable vs. fluctuating). 5. These results suggest that the performance of offspring in relation to seasonal timing of emergence is highly affected by competitive interactions in Atlantic salmon. Although a match between phenology and optimal seasonal environmental conditions may be highly important for organisms depending on specific resources that are only available during a limited period of the season, such resources may be available in variable amounts year around for many organisms. For these, offspring success may to a larger degree be shaped by the timing of their hatching/birth relative to each other, and particularly so under high population densities.

Antiviral function of tilapia hepcidin 1–5 and its modulation of immune-related gene expressions against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells

Antimicrobial peptides, small cysteine-rich molecules, play vital roles in host defense mechanisms against pathogen infection. Recently, tilapia hepcidin (TH)1–5, was characterized, and its antimicrobial functions against several pathogens were reported. Herein, we investigated the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The presence of TH1-5 enhanced the survival of CHSE-214 cells infected with IPNV. Additionally, the number of plaques formed by the cytopathic effect of IPNV in CHSE-214 cells decreased when IPNV was preincubated with TH1-5. This observation demonstrates the antiviral function of TH1-5. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. When TH1-5 and IPNV were used to co-treat CHSE-214 cells, then cells were re-challenged with IPNV at 24 h, the cells did not survive the IPNV infection. This shows that in the absence of TH1-5, viral re-challenge killed CHSE-214 cells. In conclusion TH1-5 protected CHSE-214 cells against IPNV by direct antimicrobial and immunomodulatory functions.

Development of infectious cDNA clones of Salmonid alphavirus subtype 3

BackgroundSalmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.ResultsInfectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.ConclusionWe have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.

Color Photographic Index of Fall Chinook Salmon Embryonic Development and Accumulated Thermal Units

BackgroundKnowledge of the relationship between accumulated thermal units and developmental stages of Chinook salmon embryos can be used to determine the approximate date of egg fertilization in natural redds, thus providing insight into oviposition timing of wild salmonids. However, few studies have documented time to different developmental stages of embryonic Chinook salmon and no reference color photographs are available. The objectives of this study were to construct an index relating developmental stages of hatchery-reared fall Chinook salmon embryos to time and temperature (e.g., degree days) and provide high-quality color photographs of each identified developmental stage.Methodology/Principal FindingsFall Chinook salmon eggs were fertilized in a hatchery environment and sampled approximately every 72 h post-fertilization until 50% hatch. Known embryonic developmental features described for sockeye salmon were used to describe development of Chinook salmon embryos. A thermal sums model was used to describe the relationship between embryonic development rate and water temperature. Mean water temperature was 8.0°C (range; 3.9–11.7°C) during the study period. Nineteen stages of embryonic development were identified for fall Chinook salmon; two stages in the cleavage phase, one stage in the gastrulation phase, and sixteen stages in the organogenesis phase. The thermal sums model used in this study provided similar estimates of fall Chinook salmon embryonic development rate in water temperatures varying from 3.9–11.7°C (mean = 8°C) to those from several other studies rearing embryos in constant 8°C water temperature.Conclusions/SignificanceThe developmental index provides a reasonable description of timing to known developmental stages of Chinook salmon embryos and was useful in determining developmental stages of wild fall Chinook salmon embryos excavated from redds in the Columbia River. This index should prove useful to other researchers who wish to approximate fertilization dates of Chinook salmon eggs from natural redds, assuming the thermal history of embryos is known.

A case of the Janus monstrosity found in the chum salmon embryo

A case of the Janus monstrosity found in the chum salmon embryo

The amino terminus of the salmonid alphavirus capsid protein determines subcellular localization and inhibits cellular proliferation

Salmonid alphavirus (SAV) is the most divergent member of the family Togaviridae and constitutes a threat to farming of salmonid fish in Europe. Here, we report cloning, expression and preliminary functional analysis of the capsid protein of SAV, confirming it to be expressed as an approximately 31-kDa protein in infected cells. The protein localizes strictly to the cytoplasm in Chinook salmon embryo cells, and either to the nucleus or cytoplasm in bluegill fry cells. An expression study of full-length and different truncated versions of the SAV capsid fused to the enhanced green fluorescent protein demonstrated that the localization is independent of other viral components in both cell lines, and controlled by the N-terminal 82 aa, which include a conserved, predicted helix and a downstream positively charged region. Thus, the results suggest that the SAV capsid possesses a cell-type-dependent potential for nuclear import and export. Moreover, the SAV capsid and its N-terminal 82 aa were shown to be associated with inhibition of cellular proliferation, a hallmark of the cytopathic effect caused by SAV. These results highlight that the SAV capsid is a multifunctional protein with possible importance for pathogenesis.

Nuevas armas, nuevas lesiones

Exposure to dissolved polynuclear aromatic hydrocarbons (PAHs) from crude oil delays pink salmon (Oncorhynchus gorbuscha) embryo development, thus prolonging their susceptibility to mechanical damage (shock). Exposure also caused mortality, edema, and anemia consistent with previous studies. Hatching and yolk consumption were delayed, indicating the rate of embryonic development was slowed by PAH exposure. The net result was that exposed embryos were more susceptible to shock than normal, unexposed embryos. Susceptibility to shock was protracted by 4–6 d for more than a month in embryos exposed to exponentially declining, dissolved PAH concentrations in water passed through oiled rock; the initial total PAH concentration was 22.4 μg L−1 and the geometric mean concentration was 4.5 μg L−1 over the first 20 d. Protracted susceptibility to shock caused by exposure to PAHs dissolved from oil could potentially increase the reported incidence of mortality in oiled stream systems, such as those in Prince William Sound after the Exxon Valdez oil spill, if observers fail to discriminate between direct mortality and shock-induced mortality.

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non-virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE-214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non-virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin-D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non-virulent strains were generally serum sensitive.

Tunic morphology and viral surveillance in diseased Korean ascidians: soft tunic syndrome in the edible ascidian, Halocynthia roretzi (Drasche), in aquaculture

'Soft tunic syndrome' causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non-diseased specimens using chinook salmon embryo (CHSE-214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.

Exposure of pink salmon embryos to dissolved polynuclear aromatic hydrocarbons delays development, prolonging vulnerability to mechanical damage

Exposure to dissolved polynuclear aromatic hydrocarbons (PAHs) from crude oil delays pink salmon (Oncorhynchus gorbuscha) embryo development, thus prolonging their susceptibility to mechanical damage (shock). Exposure also caused mortality, edema, and anemia consistent with previous studies. Hatching and yolk consumption were delayed, indicating the rate of embryonic development was slowed by PAH exposure. The net result was that exposed embryos were more susceptible to shock than normal, unexposed embryos. Susceptibility to shock was protracted by 4-6d for more than a month in embryos exposed to exponentially declining, dissolved PAH concentrations in water passed through oiled rock; the initial total PAH concentration was 22.4microgL(-1) and the geometric mean concentration was 4.5microgL(-1) over the first 20d. Protracted susceptibility to shock caused by exposure to PAHs dissolved from oil could potentially increase the reported incidence of mortality in oiled stream systems, such as those in Prince William Sound after the Exxon Valdez oil spill, if observers fail to discriminate between direct mortality and shock-induced mortality.

It's Elementary: Science Buddies Bring Biology to Life

As a developmental biologist with a passion for teaching, I enjoy explaining my work on cell rearrangements in the yolk sacs of killifish embryos to audiences ranging from first-year undergraduates to professional colleagues. Becoming a parent offered new opportunities. When my daughter entered our local elementary school, I wanted to find a way to share my professional life with her teachers. My modest desire to lend a helping hand evolved into a ‘‘Science Buddies’’ program, where undergraduate science majors are matched with elementary classrooms. Conceived as a way to help enrich the local science curriculum, the program has served as a training ground for young science educators, opening the eyes of college students to the joys and challenges of primary school teaching. Volunteering in the school started with an offer to bring in animals from my marine aquaria. I packed up a ‘‘Tupperware tidepool,’’ spread newspaper on the second-grade classroom floor, and passed around sea urchins and horseshoe crabs. The kids were most interested in the basics—Are they poisonous? Where did I get them? Do horseshoe crabs taste good? My visits soon expanded beyond my daughter’s class, as other teachers stopped me in the halls or sent e-mails, asking if I could bring something to their classrooms, too. One hardy sea cucumber entertained dozens of seven-year-olds and forged a link that brought my expertise to new audiences. Once I had established myself as a local scientist who loved being in the school, I was asked to be the parent liaison for a program that supplies elementary classrooms with chilled aquaria and salmon embryos for the students to hatch and rear. After four months of stewardship, the larvae are released in watershed habitats as part of an attempt to reestablish local populations. I showed time-lapse video sequences of early fish development to the fourth-grade classes, explained how a spherical egg is transformed into a small fish-on-a-ball-of-yolk, and led discussions about the salmon life cycle. When the elementary school purchased a dissecting microscope, video camera, and monitor, the microscopy cart mostly sat in the supply closet gathering dust— not because the teachers lacked the interest in the new equipment, but because they lacked the time. During a sabbatical, I made myself available to the teachers as a microscopy tech, bringing the setup into classrooms to look at whatever the class was studying—caterpillars, rocks, flower parts, etc. This was an excellent way to meet other teachers and be introduced to the science curriculum in the different grades. We expanded the salmon project by looking at the larvae under the microscope, and I brought in zebrafish embryos so the students could see much earlier stages of fish development. The first time the students saw a beating heart and blood flowing through the yolk sac of the transparent zebrafish embryos they were awestruck. They looked at the veins on the inner side of their wrists, felt their own heartbeat, and made connections between what they saw under the microscope to their own circulatory system. To ensure that these hands-on lessons would continue after my sabbatical ended, I started Science Buddies in 2005. I asked for volunteers among my undergraduate students and had them fill out a written application, looking for talented students who also had experience working with

Reference genes evaluated for use in infectious pancreatic necrosis virus real-time RT-qPCR assay applied during different stages of an infection

The stability of 6 reference genes, 18S, β-actin, RPS20, eEF1α, G6PDH and GAPDH, was examined in tissues from Atlantic salmon ( Salmo salar) and Chinook salmon embryo cells (CHSE-214). The main objective of this study was to determine the most suitable reference genes for use for the normalisation of data in quantitative real-time RT-qPCR assays conducted on infected tissues. The tissue samples selected for analysis were taken from head kidney and pylorus and collected at different time points during a challenge experiment with infectious pancreatic necrosis virus (IPNV). The stability of some of the reference genes was also studied in infected CHSE-214 cells. The ranking of the genes examined was carried out using the geNorm program. This program determines the most stable genes from a set of genes tested in a given cDNA sample. The stability of the reference genes varied in different tissues and in the cell line at different stages of infection with IPNV. This study demonstrated that tissue-specific combinations of reference genes must be used to normalise real time data for use for the quantitation of IPNV.

The winter ulcer bacterium Moritella viscosa demonstrates adhesion and cytotoxicity in a fish cell model

Moritella viscosa is considered the main aetiological agent of ‘winter ulcer’ disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7–8 °C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 °C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 °C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 °C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 °C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.

Propagation and isolation of ranaviruses in cell culture

The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28 °C for two weeks. BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells consistently produced lower titers than the other cell lines at all temperatures. The optimal temperature for propagating the isolates collectively to high titers in vivo was 24 °C. Additionally, three established methods for re-isolation of virus from EHNV-infected organ material were compared. Challenged fish were sampled twice weekly and 7 organs were processed separately according to the three methods. Samples incubated on BF-2 cells at 22 °C for 2 weeks + 1 week sub-cultivation (method 1) provided more positive results than the other 2 methods and when using the EPC cell line. Virus was most frequently isolated from the kidney, followed by brain, muscle, heart, liver, gills and lastly spleen.

Effects of fine sediment accumulation on the redd environment and the survival rate of masu salmon (Oncorhynchus masou) embryos

In recent years, fine sediment, produced by run-off associated with forestry activity and agricultural development that accumulates on riverbeds, has exerted a deleterious influence on lotic ecosystems. This study examined the Oroennukibetsu River, a tributary of the Nukibetsu River, which has been affected by high loads of suspended sediments. Effects of accumulation of fine sediment on the survival rate of masu salmon embryo and also on the redd environment (permeability and intragravel dissolved oxygen concentration) were quantified through a field experiment. Results show that the interchange of DO between intragravel and surface water was not affected directly by permeability or the accumulated fine sediment and that intragravel flow rates can be an important factor controlling embryo survival. A decrease in permeability associated with accumulation of fine sediment lowered the survival rate of embryos by suffocation because the flux of DO that should be supplied to the embryo was severely limited. This situation might be created by the combined effects of an accumulation of fine sediment on the redd and a low DO concentration in the surface water because the DO concentration almost coincided with the intragravel DO.

Temporal patterns of cold‐induced membrane restructuring in fish cells.

The cells of many animals compensate for altered environmental temperature by changing the phospholipid composition of their membranes. We monitored in fish cells the amounts of mRNA coding for some of the enzymes responsible for making these changes while the cells were exposed to reduced temperature. Chinook salmon embryo cells cultured at 20ºC were transferred to 10ºC for different times before total mRNA was extracted. Primers of our design were used to probe cDNA copies of the mRNA using PCR. PCR products were separated by agarose gel electrophoresis and digitally recorded. We found that mRNAs for the delta‐6 and delta‐9 desaturases were detectable at 20ºC and their quantities increased after 24 h, and again after 48 h, of cold exposure. The amount of message for glycerol‐3‐phosphate acyltransterase was low at 20ºC and remained low until it dramatically increased after 10 days at 10ºC. mRNAs for other enzymes showed other patterns of expression. Overall our data suggest that the metabolic adjustments that restructure membrane phospholipids in response to cold are activated at different times and persist for different intervals. It is likely they occur with a well defined temporal hierarchy. This work was supported by Undergraduate Research Grants from the Henson School of Science and Technology at Salisbury University and by a Guerrieri Summer Research Award.

ISA virus in Chile: evidence of vertical transmission

Infectious salmon anaemia virus (ISAV), genus Isavirus (family Orthomyxoviridae), is present in all large salmon (Salmo salar)-producing countries around the North Atlantic. The target species for this virus are members of the genus Salmo, but the virus may also replicate in other salmonids introduced to the North Atlantic (Oncorhychus spp.). Existing ISA virus isolates can be divided into two major genotypes, a North American (NA) and a European (EU) genotype, based on phylogenetic analysis of the genome. The EU genotype can be subdivided into several highly supported clades based on analysis of segments 5 (fusion protein gene) and 6 (hemagglutinin-esterase gene). In 1999 an ISA virus belonging to the NA genotype was isolated from Coho salmon in Chile, and in 2007 the first outbreaks of ISA in farmed Atlantic salmon was observed. Several salmon farms in Chile were affected by the disease in 2007, and even more farms in 2008. In this study, ISA virus has been isolated from salmon in a marine farm suffering an outbreak of the disease in 2008 and from smolts with no signs of ISA in a fresh water lake. Sequencing of the partial genome of these ISA viruses, followed by phylogenetic analysis including genome sequences from members of the NA and EU genotypes, showed that the Chilean ISA virus belongs to the EU genotype. The Chilean ISA virus groups in a clade with exclusively Norwegian ISA viruses, where one of these isolates was obtained from a Norwegian brood stock population. All salmonid species in the southern hemisphere have been introduced from Europe and North America. The absence of natural hosts for ISA viruses in Chile excludes the possibility of natural reservoirs in this country, and the close relationship between contemporary ISA virus strains from farmed Atlantic salmon in Chile and Norway suggest a recent transmission from Norway to Chile. Norway export large amounts of Atlantic salmon embryos every year to Chile; hence, the best explanation for the Norwegian ISA virus in Chile is transmission via these embryos, i.e. vertical or transgenerational transmission. This supports other studies showing that the ISA virus can be transmitted vertically.