Sample displacement chromatography of Atlantic Salmon (Salmo salar) thrombin

A modified method of sample displacement chromatography (SDC) was used to purify active salmon thrombin on a heparin-coupled matrix to near homogeneity in milligram amounts from 117 ml plasma. This was achieved by combining a low-pressure multi-column affinity chromatography system with non-homogenous sample application in the order of increasing affinity to Heparin Sepharose. The results suggest that this modified method could be useful in protein purification. Some characteristics of salmon thrombin are presented.     

Proteomic Analysis of Chinook Salmon (Oncorhynchus tshawytscha) Ovarian Fluid

The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.     

Individual Monitoring of Immune Response in Atlantic Salmon Salmo salar following Experimental Infection with Infectious Salmon Anaemia Virus (ISAV)

Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m³ tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. “In-tank” anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and γIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or γ IFN could be detected. These results and the advantages of this approach are discussed.     

Tissue Distribution of 14C-Diflubenzuron in Atlantic Salmon (Salmo salar)

Diflubenzuron is a potent inhibitor of chitin synthesis, with potential use against salmon lice infestations. The absorption, distribution and elimination of the substance in Atlantic salmon was examined after a single, oral dose of 75 mg/kg body weight. The kinetic properties were studied by whole-body autoradiography, liquid scintillation counting and thin layer chromatography, using a 14C-labelled isotope of the substance. The drug was poorly absorbed from the intestine, but reached a concentration of more than 4 µg/g in the mucus layer of the skin 2 days after administration. If maintained for several days, this concentration is probably sufficient to control all moulting stages of sea lice in Atlantic salmon. The main route of excretion was via the bile.     

Reproduction of Pacific Salmon (Oncorhynchus) in Rivers Flowing into Terpeniya Bay (Sakhalin Island)

Journal of Ichthyology - The conditions for the reproduction of Pacific salmon of the Oncorhynchus genus in rivers flowing into Terpeniya Bay were assessed. The differences between the rivers of...     

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Prominent Orcadian Absorption of Intranasal Salmon Calcitonin (SCT) in Healthy Subjects

In young healthy subjects salmon calcitonin (SCT), intranasally administered, increased in serum as a function of the drug administration time. The serum concentration of a 400 IU SCT dose monitored 10 min after dosing was statistically more significant when inhaled at 0000 than at other, more conventional, administration times (morning or evening). Following dosing at certain times during the day, the serum SCT was less or even questionable with the dose and under the study conditions selected. Dosing without consideration of timing may lead to reduced effect or lack of effect or perhaps ambiguity or controversy regarding the possible circumstance of a “non-absorbent subject”. The circadian frequency appears to be a critical determinant of intranasal SCT absorption suggesting administration time to be an important factor in the cost/benefit ratio without the unpleasant side effects sometimes experienced through parenteral routes.     

Cataract Development in Atlantic Salmon (Salmo salar L) in Fresh Water

Irreversible bilateral cataracts were diagnosed by slit-lamp biomicroscopy in 178 of 200 farm-raised Atlantic salmon (Salmo salar L) fed a standard diet over a five-month period. Initial changes were anterior polar opacities, progressing to involve both the anterior and posterior cortex before changes in the lens nucleus were seen. The lens changes were recorded and given scores according to the severity of the cataracts. At each of 3 samplings, after 2, 4 and 5 months, 200 fish were measured, weighed and examined by slit-lamp biomicroscopy. At all 3 samplings, there was a significant correlation between body length and both cataract incidence and cataract severity. There was also a significant correlation between body weight and cataract incidence and severity for the 2 last samplings. There was a significant correlation between K-factor as a measure of the shape of the fish, and both cataract incidence and severity, at all 3 samplings. Evaluation of specific growth rate in the periods between the examinations showed that the rapidly-growing fish were most susceptible to cataract formation. After cataract developed, however, the growth rate slowed. Follow-up examination of severely affected fish 3 months after transfer to sea water showed a normal cortical zone in the periphery of the lens in 24 out of 28 fish.     

N-Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N-terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS-PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N-terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N-terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.     

Prominent Orcadian Absorption of Intranasal Salmon Calcitonin (SCT) in Healthy Subjects

In young healthy subjects salmon calcitonin (SCT), intranasally administered, increased in serum as a function of the drug administration time. The serum concentration of a 400 IU SCT dose monitored 10 min after dosing was statistically more significant when inhaled at 0000 than at other, more conventional, administration times (morning or evening). Following dosing at certain times during the day, the serum SCT was less or even questionable with the dose and under the study conditions selected. Dosing without consideration of timing may lead to reduced effect or lack of effect or perhaps ambiguity or controversy regarding the possible circumstance of a “non-absorbent subject”. The circadian frequency appears to be a critical determinant of intranasal SCT absorption suggesting administration time to be an important factor in the cost/benefit ratio without the unpleasant side effects sometimes experienced through parenteral routes.     

Dispersal Potential is Affected by Growth-Hormone Transgenesis in Coho Salmon (Oncorhynchus kisutch)

Potential ecological consequences on the natural environment of fast‐growing transgenic fish with elevated intrinsic growth rates is an important question should such fish be allowed in commercial production systems. One trait that will strongly affect the spatial extent of consequences is the propensity of transgenic fish to disperse. We addressed this question in three experiments using different measures of spatial dispersal where we compared very young genotypically wild coho salmon with transgenic conspecifics in terms of: (i) group cohesion, (ii) exploratory behavior, and (iii) up‐ and downstream movements. Transgenic fish were more loosely aggregated, more likely to explore, and less likely to swim upstream, but equally likely to disperse downstream compared with genetically wild fish. These results show that dispersal behavior has been affected by transgenesis and that transgenic fish therefore may venture into habitats previously not used by wild fish. Given the importance of dispersal in ecological risk‐assessment of transgenic fish, continued work within this area is warranted and experimental habitats should mimic the potential receiving natural habitats to which transgenic fish are likely to escape or be released to.     

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis.     

Inheritance of Alpha-Glycerophosphate Dehydrogenase in the Pink Salmon, ONCORHYNCHUS GORBUSCHA (Walbaum)

Genetic crosses of alpha-glycerophosphate dehydrogenase (alpha-GPDH) phenotypes in the pink salmon, Oncorhynchus gorbuscha (Walbaum), reveal that this enzyme is encoded by a single locus with two codominant alleles (Slow and Fast). The significance of single gene control of alpha-GPDH is discussed in relation to the purported tetraploid nature of the salmonids.     

Coho Salmon (Oncorhynchus kisutch) Prefer and Are Less Aggressive in Darker Environments

Fish are capable of excellent vision and can be profoundly influenced by the visual properties of their environment. Ambient colours have been found to affect growth, survival, aggression and reproduction, but the effect of background darkness (i.e., the darkness vs. lightness of the background) on preference and aggression has not been evaluated systematically. One-hundred Coho salmon (Oncorhynchus kisutch), a species that is increasing in popularity in aquaculture, were randomly assigned to 10 tanks. Using a Latin-square design, every tank was bisected to allow fish in each tank to choose between all the following colour choices (8 choices in total): black vs. white, light grey, dark grey, and a mixed dark grey/black pattern, as well as industry-standard blue vs. white, light grey, dark grey, and black. Fish showed a strong preference for black backgrounds over all other options (p < 0.01). Across tests, preference strength increased with background darkness (p < 0.0001). Moreover, having darker backgrounds in the environment resulted in less aggressive behaviour throughout the tank (p < 0.0001). These results provide the first evidence that darker tanks are preferred by and decrease aggression in salmonids, which points to the welfare benefits of housing farmed salmon in enclosures containing dark backgrounds.     

硬头鳟与溪红点鲑杂交三倍体育种技术的初步研究

为了获得同时具有杂交及三倍体优势的鲑鳟鱼苗种,开展了利用6-二甲基氨基嘌呤(6-DMAP)诱导硬头鳟(Oncorhynchus mykiss)、溪红点鲑(Salvelinus fontinalis)正反杂交受精卵的实验。杂交结果,溪红点鲑(♀)×硬头鳟(♂)实验组卵子在受精10 min后全部死亡,而硬头鳟(♀)×溪红点鲑(♂)组的受精卵正常,可以进行6-DMAP三倍体诱导实验。6-DMAP诱导受精卵结果,起始诱导时间在12~15 min,受精卵的发眼率最高,为75.87%~76.64%,在5个起始诱导时间梯度内,受精卵的孵化率在86.44%~90.31%之间,苗种三倍体率在92.45%~95.55%之间,差异不显著(P0.05)。诱导持续时间为10~15 min时,受精卵发眼率达到最高,为74.28%~76.81%,在5个诱导持续时间梯度内,受精卵孵化率在87.28%~90.24%之间,苗种三倍体率在93.67%~96.25%之间,变化不显著(P0.05)。药物诱导浓度为120~150 mg/L时,受精卵的发眼率达到最高,为73.57%~76.27%,在5个不同的药物浓度梯度内,受精卵孵化率在88.57%~90.03%之间,苗种三倍体率在92.56%~96.38%之间,变化不显著(P0.05)。结果表明,利用6-DMAP诱导受精卵制备杂交三倍体苗种的方法是可行的,实验最佳方法为硬头鳟(♀)×溪红点鲑(♂)卵子受精后12~15 min内,利用浓度为120~150 mg/L的6-DMAP溶液诱导,持续时间为10~15 min,然后放入孵化桶内正常孵化,获得苗种的三倍体率可达92.56%~96.38%,可满足正常生产的需要。     

Fate of radioactivity in Atlantic Salmon (Salmo salar) following intragastric administration of (methyl-14C)-trichlorfon

The antiparasitic drug Neguvon® (Bayer), with the active component trichlorfon (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate), is extensively used in fish farms in Norway. The fate of (methyl-¹⁴C)-trichlorfon was tested in Atlantic salmon (Salmo salar) by liquid scintillation counting at day 1, 4, 7, 14, and 30 post administration, and by autoradiography on selected organs 24 h after administration. The remaining radioactivity was found to be high compared to earlier measurements of the trichlor)fon content made by gas chromatography (Brandal 1977). The grains in autoradiographic preparations of muscle were unevenly distributed both in the muscle as a whole and within muscle fibers. In liver the grains were evenly distributed, but were absent from fat vacuoles. The study indicate that the radioactive residues in salmon muscle do not represent trichlorfon or its derivative dichlorvos (0,0-dimethyl-0-(2,2-dichlorovinyl)-phosphate), but rather hydrophilic metabo)lites of these compounds.     

Forensic identification of endangered Chinook Salmon (Oncorhynchus tshawytscha) using a multilocus SNP assay

Upper Columbia River spring-run Chinook salmon are listed as endangered under the Endangered Species Act (ESA). Forensic genetic analyses are needed to assist the National Oceanic and Atmospheric Administration Fisheries Office of Law Enforcement (NOAA Fisheries OLE) during criminal investigations of ESA take violations. Previous genetic studies using allozyme markers have demonstrated that the spring-run and summer-run of Chinook salmon in the Upper Columbia River are genetically differentiated. Because many of the carcasses collected as forensic evidence are of compromised quality, we have developed a PCR based assay to distinguish between the spring-run and the summer-run Chinook salmon in the upper Columbia River Basin. A total of 347 Chinook salmon samples from the upper Columbia River in Washington State were surveyed for single nucleotide polymorphisms (SNPs) using PCR-RFLP at two nuclear genetic loci (Somatolactin and Cytochrome p450A), and a mitochondrial locus (Cytochrome Oxidase III/ND3). We found near fixed differences in our SNP baseline between the summer-run and the spring-run Chinook salmon in the upper Columbia River at these loci enabling us to assign individuals to the most likely population of origin with a high degree of accuracy.     

N‐Terminal Sequence and Main Characteristics of Atlantic Salmon (Salmo salar) Albumin

Abstract

Atlantic salmon (Salmo salar) serum albumin was purified from plasma and its N‐terminal sequence determined. Atlantic salmon albumin is the predominant plasma protein, negatively charged, at pH 8.6. Albumin was purified to >95% purity which yielded a single band on SDS‐PAGE and agarose gel electrophoresis. The molecular weight of the purified albumin was approximately 6,5 kDa. The N‐terminal sequence of Atlantic chinook salmon albumin was consistent with that predicted from its previously determined cDNA sequence and was identical to that of salmon (Oncorhynchus tshawytscha) albumin through the first 15 residues. However, the fact that the actual N‐terminus was different from that predicted from cDNA sequence indicates that Atlantic salmon albumin, like chinook salmon albumin, lacks a propeptide.