O-acetylation of sialic acids in N-glycans of Atlantic salmon (Salmo salar) serum is altered by handling stress

O-acetylation is one of the major modifications of sialic acids that significantly alters biological properties of the parent molecule. These O-acetylated forms are components of the cellular membrane and can affect physiological and pathological responses. Understanding the role of N-glycans in physiology is of increasing relevance to cellular biologists in various disciplines who study glycoproteomics yet lack information regarding the function of the attached glycans. It is well known that stress may decrease immune function in fish; however, there are only few suitable biomarkers available to monitor the physiological responses under the stress conditions. This study is the first report on the effect of stress on the profile of O-acetylation of sialic acids in fish serum. In order to preserve the relevant structural characteristics as much as possible, native N-glycans were directly analyzed using CE-MS. We have characterized the N-glycans in serum of salmon (Salmo salar) exposed to long-term handling stress (15 s out of the water, daily for 4 wk) and compared with the results obtained from sera of control fish. The results indicated that major N-glycans in salmon serum contained mono-acetylated sialic acids (83%), and that the O-acetylation pattern of sialic acids could be altered by long-term stress.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Simple, automated, high resolution mass spectrometry method to determine the disulfide bond and glycosylation patterns of a complex protein: subgroup A avian sarcoma and leukosis virus envelope glycoprotein

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.     

Effects of hydropeaking on the spawning behaviour of Atlantic salmon Salmo salar and brown trout Salmo trutta

An in situ camera set‐up was used to study the spawning activity of Atlantic salmon Salmo salar and brown trout Salmo trutta throughout two consecutive seasons in a spawning area affected by hydropower‐related pulse flows due to hydropeaking. The purpose was to test whether the flow variation discouraged spawning in shallow areas or motivated spawning into areas with elevated risk of incubation mortality. There were more S. salar observed on the spawning ground during days with high discharge. The presence of S. salar in the spawning grounds was not affected by the hydropeaking cycles of the preceding night. Female S. salar were observed preparing nests within the first hour after water discharge had increased to levels suitable for spawning. In contrast, the number of S. trutta was not correlated with flow and nest preparation was also observed at a discharge corresponding to the lowest discharge levels during a hydropeaking cycle. Survival was generally high in nests excavated the following winter, with only 5·4% suffering mortality due to dewatering. The results suggest that S. salar may respond rapidly to variable‐flow conditions and utilize short windows with suitable flows for spawning. Smaller S. trutta may utilize low‐flow conditions to spawn in areas that are not habitable by larger S. salar during low flow.     

Effects of temperature and feed intake on astaxanthin digestibility and metabolism in Atlantic salmon, Salmo salar

The effects of feed intake, growth rate and temperature (8 and 12 °C) on apparent digestibility coefficients (ADC), blood uptake of individual astaxanthin E/Z isomers and metabolism of astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) were determined in Atlantic salmon. Accumulation of idoxanthin (3,4,3′-trihydroxy-β,β-carotene-4-one) in plasma was used to indicate metabolic transformation of astaxanthin. Quadruplicate groups of fish were subjected to three different treatments; one treatment was kept at 12 °C and fed to satiation. Another treatment kept at 12 °C was pair-fed with fish fed to satiation at 8 °C, resulting in a restricted feeding regime for the former treatment. After 2 months of feeding, the fish were fed a single meal containing ballotini glass beads to determine individual feed intake and Y₂O₃ as an inert marker to determine ADCs. The faeces samples were pooled into 6 categories according to individual meal size (range 0.2–1.5% of body weight) and the ADCs for different meal sizes were determined. ADCs of astaxanthin ranged from 20% to 60% but were not significantly correlated with meal size. However, fish kept at 12 °C had approximately 10% higher ADC than fish kept at 8 °C (p = 0.032). Growth rate and plasma astaxanthin concentration were higher at higher temperature and higher ration. Plasma concentration of idoxanthin was not affected by temperature or by meal size. The incidence of fin erosion and non-feeding individuals was significantly higher among fish fed a restricted ration indicating more aggressive interactions. Fish with visible fin damage had a tendency for having higher idoxanthin content in plasma than fish without noticeable fin damage. It is concluded that temperature but not individual meal size affect ADC of astaxanthin, whereas both influence plasma astaxanthin levels and may therefore affect the efficiency of astaxanthin utilization.     

Effects of discharge and local density on the growth of juvenile Atlantic salmon Salmo salar

The study explored the combined effects of density, physical habitat and different discharge levels on the growth of juvenile Atlantic salmon Salmo salar in artificial streams, by manipulating flow during both summer and winter conditions. Growth was high during all four summer trials and increased linearly with discharge and mean velocity. Differences in fish densities (fish m^?3) due to differences in stream volume explained a similar proportion of the variation in mean growth among discharge treatments. Within streams, the fish aggregated in areas of larger sediment size, where shelters were probably abundant, while growth decreased with increasing densities. Fish appeared to favour the availability of shelter over maximization of growth. Mean growth was negative during all winter trials and did not vary among discharge treatments. These results suggest that increased fish densities are a major cause of reduced summer growth at low discharge, and that habitat‐mediated density differences explain the majority of the growth variation across habitat conditions both during summer and winter.     

Effects of storage time on the motility,mortality and calcium levels of Atlantic salmon Salmo salar spermatozoa

This study estimates spermatozoa mortality, morphology, motility and intracellular calcium levels in Atlantic salmon Salmo salar milt after prolonged storage. Milt samples were preserved at 4° C for 25 days and then evaluated for mortality. Motility remained high for the first 3 days and the mortality was low during the first 5 days of storage. A decrease of >50% in calcium content was observed after 5 days of storage. When spermatozoa were activated, calcium levels increased >200% in relative fluorescence units (RFU); this rate of increase was lost when the samples were stored for extended periods of time and was only partially manifested in a zero calcium solution. The results suggest that in vitro storage of S. salar spermatozoa at 4° C for a period of 3 days preserves motility and limits mortality to levels similar to those of fresh spermatozoa. This method also maintains intracellular calcium storage critical for spermatozoa performance.     

Chronic effects of low pH on length and weight of Atlantic salmon,Salmo salar

Synopsis Measurements were made on Atlantic salmon from five sources in New England. Fish were reared at pH 7.0 and 5.2 from hatch to 199, 270 or 440 days. Length and weight were smaller in Atlantic salmon, Salmo salar, that were continuously exposed to low pH in the laboratory. Length and weight in neutral pH water varied among fish from the different sources. Fish from the Green Lake, Craig Brook, and Tunison sources were not significantly different in their lengths over time. Cortland and landlocked Atlantic salmon were not significantly different from one another but were significantly different from the other three sources. At pH 5.2, there were no significant differences in length between fish from any of the five sources. The Cortland salmon were significantly different from the other four sources in respect to their weight over time at neutral pH. At pH 5.2, there were no significant differences between any of the five sources.Cooperators are the U.S. Fish and Wildlife Service, the Maine Department of Inland Fisheries and Wildlife, the University of Maine, and the Wildlife Management Institute     

Atlantic salmon (Salmo salar) of the Pigou and Bouleau Rivers,Quebec

Synopsis Juvenile Atlantic salmon of the Pigou and Bouleau Rivers, Quebec, were studied during the summer of 1972 and 1973. Growth increments for Pigou salmon averaged 36–50 mm yr^–1 and smolts averaged 2.9 yrs old. Bouleau salmon grew slower (26–41 mm yr^–1) and became smolts at a slightly older mean age (3.1 yrs). Mature male parr were more abundant in all age-groups from the Plgou River, possibly because of the faster growth. In terms of management, the Pigou River has little potential for increased salmon production because of limited habitat. On the other hand, the Bouleau River has considerable potential, and salmon production could be increased if the upper reaches were made accessible to migrating salmon.Contribution number 36 of the Matamek Research Station, Woods Hole Oceanographic Institute.     

Effects of habitat on individual reproductive success of mature male parr of Atlantic salmon Salmo salar

The effect of the presence of stone blocks in the spawning habitat on the reproductive success of mature male parr of Atlantic salmon Salmo salar of various sizes and ages was tested in an artificial channel. Shelters allowed smaller individuals to contribute to egg fertilization as much as large parr, suggesting that the size-based dominance observed in a shelterless habitat was not maintained in a more complex habitat.     

Effects of elevated nitrate concentration on early thyroid morphology in Atlantic salmon (Salmo salar Linnaeus, 1758)

A study was conducted to determine if exposure to elevated nitrate (NO₃^‐)(3.76 ± 0.07 and 93.15 ± 2.79) during embryonic development influences the morphology of thyroid tissue in newly hatched Atlantic salmon, Salmo salar. Digital three‐dimensional imaging of thyroid follicles in S. salar showed that the thyroid tissue in newly hatched alevin consisted mainly of spherical follicles dispersed throughout the lower jaw in close proximity to the ventral aorta and other blood vessels. With these digital models, the number of follicles, average volume per follicle, and total luminal volume were calculated for alevin exposed to well water or 93.15 mg L^?1 NO₃‐N during embryogenesis. There were no statistically significant differences in any of these parameters, suggesting that the normal thyroid development of S. salar could be relatively insensitive to excess environmental NO₃^‐. These results are promising for the culture of Atlantic salmon embryos in recirculating aquaculture systems that naturally accumulate NO₃^‐ as a product of nitrification.     

Effects of pre- and post-prandial starvation on meal size and evacuation rate of juvenile Atlantic salmon, Salmo salar L.

After a pre-prandial period of starvation or feeding with unlabelled food, 0+ salmon parr (0.8–11.7 g) were fed a test meal of iron particle labelled food and subsequently were again either starved or fed unlabelled food. The quantity of labelled food consumed and the evacuation rate was determined by serial radiographs. In fish of all sizes, pre-prandial starvation causes a larger test meal (as a percentage of body weight) to be consumed when compared to pre-prandially fed fish. In addition, pre-prandial starvation results in relatively larger meals as a percentage of body weight being taken by smaller compared to larger fish. This result was not evident for pre-prandially fed fish. Evacuation rate was unrelated to body size irrespective of feeding history. Post-prandial starvation decreased evacuation rate but this effect was inversely related to the quantity of food consumed. Larger meals were not evacuated differently from smaller meals if feeding occurred post-prandially, irrespective of pre-prandial starvation.     

Links between patterns of marine growth and survival of Atlantic salmon Salmo salar, L.

The hypothesis that marine survival of Atlantic salmon Salmo Salar is linked to marine growth was explored by using inter-circuli distances and total numbers of circuli existing on scales from a population monitored over nearly four decades. The results suggest that marine growth controls survival, particularly during the late summer and early winter of the first year at sea. Recruitment is strongly linked to growth, described as the total number of circuli, but not to inter-circuli distances. This highlights the potential of patterns of circuli number to be considered as proxies for growth. Indications that hatchery populations might be subject to other mortality events, in addition to those experienced by wild populations, are also presented.     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

The effect of the size of Atlantic salmon, Salmo salar L., eggs on embryos and alevins

A marked diversity in the durations of river and sea existence and the frequency in spawning is shown in the samples of Atlantic salmon females, which were collected over six years. The data are presented on the size and weight of eggs and their dependence on age, and size of females and the time spent at sea. A correlation was established between the size of eggs of embryos and alevins.     

Water chemistry and its effects on the physiology and survival of Atlantic salmon Salmo salar smolts

The physiological effects of episodic pH fluctuations on Atlantic salmon Salmo salar smolts in eastern Maine, U.S.A., were investigated. During this study, S. salar smolts were exposed to ambient stream-water chemistry conditions at nine sites in four catchments for 3 and 6 day intervals during the spring S. salar smolt migration period. Plasma chloride, plasma glucose, gill aluminium and gill Na(+)- and K(+)-ATPase levels in S. salar smolts were assessed in relation to ambient stream-water chemistry during this migration period. Changes in both plasma chloride and plasma glucose levels of S. salar smolts were strongly correlated with stream pH, and S. salar smolt mortality occurred in one study site with ambient stream pH between 5·6 and 5·8 during the study period. The findings from this study suggest that physiological effects on S. salar smolts are strongly correlated with stream pH and that in rivers and streams with low dissolved organic carbon (DOC) concentrations the threshold for physiological effects and mortality probably occurs at a higher pH and shorter exposure period than in rivers with higher DOC. Additionally, whenever an acidification event in which pH drops below 5·9 coincides with S. salar smolt migration in eastern Maine rivers, there is potential for a significant reduction in plasma ions of S. salar smolts.     

Mitochondrial DNA and allozyme analysis of Atlantic salmon, Salmo salar L., in England and Wales

Allozyme variation was examined in 429 Atlantic salmon, from seven sampling sites in England and Wales. Contingency tests for genetic homogeneity using three diagnostic loci revealed no significant differences among populations from the north-east and north-west of the U.K., but detected significant aspartate aminotransferase (AAT) differences between these samples and those from the R. Itchen in Hampshire (southern England). Mitochondrial DNA variation was analysed in 40 salmon from five spawning sites in the R. Itchen. Eight restriction endonucleases were used, of which three (AVAII, HAE III and HINFI) revealed polymorphisms. Six matriarchal lineages were discriminated. Two of the six mtDNA types observed were site-specific. These results suggest a reasonable expectation of discriminating spawning aggregations of Atlantic salmon.     

Observations of the biology and parasites of postsmolt Atlantic salmon, Salmo salar, from the Norwegian sea

The main food items of postsmolt Atlantic salmon caught in the Norwegian sea during summer 1991 were Parathemisto spp. and herring ( Chipca harengus ). The most prevalent parasite species were Chloromyxion sp., Apatemon sp., H. adunction, A simplex and I. salmonis .