Effect of environmental temperature on biosynthesis of liver phosphatidyl-choline in the trout (Salmo gairdnerii)

1. 1.The changes in fatty acid composition of trout liver phosphatidyl-choline, when the environmental temperature was increased, resulted in modifications in the amounts of the different molecular species.

2. 2.In vivo, incorporation of ³²P occurred more rapidly in the molecular species with high degree of unsaturation.Temperature acclimation did not modify the relative proportions of label shown in the five subfractions.

3. 3.In vitro incorporation of ¹⁴C-methyl-choline also occurred more rapidly in the most unsaturated molecular species. However, when the incubation temperature was raised, the proportion of label decreased in the unsaturated fractions.

Hemoglobin components from trout (Salmo irideus): determination of their peroxidative activity

The peroxidative activity of trout hemoglobins, HbI and HbIV, which differ in their conformation, was compared with that of HbA. Artificial substrates (guaiacol and dopamine) and more physiological substrates such as model lipid membranes containing unsaturated fatty acids were used. The results indicate that all the hemoglobin molecules assayed show different levels of peroxidative activity. The capability to act as peroxidases is greater in HbIV than in HbI and HbA. In contrast, native globins did not show peroxidase activity. The different peroxidative activity of the Hbs is discussed in relation to stability both vs. protein oxidation and protein dissociation. The results confirm the view that hemoglobin may be of importance in establishing the life span of the erythrocyte itself.     

The entrainment and gating of the endogenous circannual rhythm of reproduction in the female rainbow trout (Salmo gairdneri)

Summary The endogenous circannual rhythm of maturation in the female rainbow trout and associated changes in serum oestradiol-17 and vitellogenin (as calcium) can be entrained by abrupt changes in the photoperiod. Groups of virgin fish (which naturally spawn in December) maintained under LD 186 from mid-January (Year 1), and then subjected to a reduction to LD 618 either on 1st March (Group A), 1st April (Group B), 1st May (Group C) or 1st June (Group D), commenced spawning on 31st July, 13th and 30th August, and the 16th September, respectively. Fish maintained on LD 186 throughout the experiment (Group E) commenced spawning on 10th October. These advances in the timing of spawning can be described in the form of a phase-response curve analogous to the entrainment behaviour of circadian oscillators. It is concluded that under natural conditions the annual change in photoperiod serves to continuously entrain the circannual clock thus ensuring that maturation and spawning occur at the optimal season for the survival of the species.In Groups A, B, C and D, respectively, 74%, 48%, 23% and 8% of the fish failed to mature in Year 1. Maintaining a sample of both maturing and non-maturing trout under constant LD 618 for a further year resulted in all the fish spawning in August of Year 2, approximately one year after the spawning members of this group matured in Year 1. It is proposed that the reduction from LD 186 to LD 618 phase-advanced a circannual timing mechanism in all the fish which then free-ran; this advance was only overtly expressed by the earlier spawning of a certain percentage of the fish in Year 1, but in all the fish in Year 2. These results indicate that the internal timing mechanism can be dissociated from the neuroendocrine mechanisms controlling maturation and thus it can be considered as an autonomous circannual clock. The relationship between the timing of the reduction in photoperiod and the percentage of virgin fish attaining maturation can be explained by a gating model. It is hypothesized that the energetically demanding process of gonadal maturation is only allowed in virgin fish which have reached a certain threshold stage of development and when the clock is at a specific (gate open) phase of the circannual cycle.     

Variations in the volumes of parenchyma and stroma of the liver and in the cytology of hepatocytes are associated with gonadal stages in female Ohrid trout (Salmo letnica)

Quantitative microscopic studies of season- and breeding-related changes in the liver of farm-raised fish are very scarce, and those of wild fish populations are virtually nonexistent. Moreover, none of the available studies investigated breeding-related changes in hepatic stroma, although it is already known that changes might exist in the parenchyma. To the best of our knowledge, ours is the first study using wild adult Ohrid trout females to evaluate quantitative influences of the breeding cycle on the volumes of the two liver compartments—parenchyma and stroma. Quantitative microscopy (stereology) using light microscopy was supplemented with semi-quantitative and quantitative analyses of hepatocyte cytology to express in numbers the seasonal changes in the extent of vacuolated versus basophilic cytoplasm. The liver volume and that of each compartment changed from that at the time of pre-vitellogenesis to the end of the spawning season. The changes in total volumes of parenchyma and stroma—increasing from pre-vitellogenesis to late vitellogenesis—followed the changes in total liver volume. Despite all the changes in total volumes, no differences existed in the relative proportions between the two major compartments. After spawning, the stromal volume continued to increase while that of the parenchyma reduced despite no detectable statistically significant impacts on the liver volume. Changes in both the liver and parenchyma volumes were correlated with the gonadosomatic index and plasma oestradiol levels. With this study, we first establish that fish liver remodelling might also occur in the stroma. When comparing Ohrid trout with other fish species, we noted interspecies similarities and differences with regard to the hepatocyte cytology during the breeding cycle. In our study, the semi-quantitative and stereological studies revealed that, from pre-vitellogenesis to post-spawning, although cytoplasmic vacuolation of the hepatocytes decreased, the basophilia increased. Overall, these cytological changes were only partially in agreement with the data from other trout studies. We believe that this is because of intrinsic interspecies differences in association with natural conditions. Thus, establishing normal patterns is crucial, at least for flagship species, namely to support interpretation of histopathological changes in biomonitoring.     

Time course of osmotic adaptation and gill energetics of rainbow trout (Salmo gairdneri R.) following abrupt changes in external salinity

Summary The physiological and biochemical responses of rainbow trout (mean weight 250 g) to abrupt increases in salinity have been investigated. An initial crisis period lasting about 30 h was characterized by an increase in plasma and muscle ions, a rapid gill dehydration and a pronounced acidosis following a transient alkalosis. Mortality was low during this period. During the following days, gradual changes resulted in new steady state levels for most parameters examined.Analysis of adenylate pool (ATP, ATP/ADP ratio and energy charge) in the gills demonstrated an increased energy demand exhibiting two phases (4 h and 3 days), and a return to freshwater values. The gill respiration rate was constant during 3 days in sea water and decreased slightly later on. It was not influenced during the reverse transfer of seawater adapted fish into fresh water at the level of either isolated gills or perfused heads.     

Incorporation of 1-14C-acetate into fatty acids and sterols by isolated hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri)

Summary Rates of 1-¹⁴C-acetate incorporation into specific fatty acids and sterol fractions were determined at assay temperatures of 5°C and 20°C in hepatocytes isolated from warm (20°C)- and cold (5°C)- acclimated rainbow trout (Salmo gairdneri). Rates of sterol lipogenesis were 2.5- to 3-fold higher in hepatocytes from cold-acclimated trout. Rates of acetate oxidation and of total fatty acid lipogenesis did not differ significantly between acclimation groups. Fatty acid compositions did not change significantly during the experiment (9–12 h), but hepatocytes from cold-acclimated trout possessed significantly higher levels of polyunsaturates and unsaturates of the linolenic acid (n-3) family, and significantly lower levels of monounsaturates than did hepatocytes from warm-acclimated animals. Hepatocytes from cold-acclimated trout channeled a larger percentage of their total acetate incorporation into unsaturated fatty acids at 5°C than at 20°C due primarily to increased recovery of acetate in polyunsaturates and monoenes at 5°C. In contrast, hepatocytes from warm-acclimated trout channeled a slightly smaller percentage of their total acetate incorporation into unsaturates at 5°C than at 20°C. Hepatocytes from warm-acclimated trout incorporated significantly more 1-¹⁴C-acetate into the unsaturated fatty acid fraction (due primarily to incorporation into the diene fraction and less importantly all other classes of unsaturates) and significantly less into the saturated fatty acid fraction than hepatocytes from cold-acclimated trout when assayed at 20°C; similar but less dramatic differences were observed at 5°C. Consequently, unsaturated/saturated ratios for acetate incorporation ranked: warm-acclimated at 20°Cwarm-acclimated at 5°Ccold-acclimated at 5°C>cold-acclimated at 20°C. These results suggest that regulation of the relative rates of unsaturated and saturated fatty acid synthesis is involved in lipid restructuringduring adaptation from one temperature regime to another, but that other mechanisms must be invoked to explain the maintenance of observed steady state differences between the fatty acid compositions of warm- and cold-acclimated trout.This work was supported by grant PCM-76-04313-AO1 from the National Science Foundation     

Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout

We investigated the effects of nutritional state on carbohydrate, lipid, and protein stores in the heart, liver, and white skeletal muscle of male and female rainbow trout. For fed animals we also partitioned glycogen into fractions based on acid solubility. Fish (10-14 months-old, ~400-500 g) were held at 14 °C and either fed (1% of body weight, every other day) or deprived of food for 14 days. Under fed conditions, glycogen was increased 54% in ventricles from males compared with females, and elevated in the liver (87%) and white muscle (70%) in sexually-maturing versus immature males. Acid soluble glycogen predominated over the acid insoluble fraction in all tissues and was similar between sexes. Food deprivation 1) selectively reduced glycogen and free glucose in male ventricles by ~30%, and 2) did not change glycogen in the liver or white muscle, or triglyceride, protein or water levels in any tissues for both sexes. These data highlight sex differences in teleost cardiac stores and the metabolism of carbohydrates, and contrast with mammals where cardiac glycogen increases during fasting and acid insoluble glycogen is a significant fraction. Increased glycogen in the hearts of male rainbow trout appears to pre-empt sex-specific cardiac growth while storage of acid soluble glycogen may reflect a novel strategy for efficient synthesis and mobilization of glycogen in fishes.     

Plasma cortisol measurements in precocious male and nonprecocious male juvenile steelhead trout (Salmo gairdneri)

Summary The mean biweekly plasma cortisol levels of juvenile nonmigratory precocious male steelhead trout,Salmo gairdneri, maintained from June through November in our laboratory under natural photoperiodic conditions ranged from 2.4–27.2 g/dl. Greatest mean cortisol occurred during the month of greatest gonadal mass, suggesting a direct interrenal-testicular relationship in this population of trout. The monthly mean plasma cortisol level of nonprecocious juvenile steelhead males captured prior to their downstream migration to the ocean from an Idaho hatchery and maintained in the laboratory simultaneously with the precocious males was low. For some months plasma cortisol was less than 0.1 g/dl. In June, however, plasma cortisol in nonprecocious males was 11.0 g/dl.     

Stress-induced expression of protein disulfide isomerase associated 3 (PDIA3) in Atlantic salmon (Salmo salar L.)

In mammals, disulfide isomerase associated 3, PDIA3, is a member of the endoplasmic reticulum (ER) stress proteins, which can be induced by oxidative stress; however, its role in relation to stress regulation is still unknown in fish. Here, we report the cloning of a coding region of PDIA3 from the Atlantic salmon. PDIA3 mRNA expression was evaluated in the liver of Atlantic salmon exposed to environmental hyperoxia stress and toxic perfluorooctane sulfonate (PFOS) exposure stress. The PDIA3 sequence contained two PDI-typical thioredoxin active sites of WCGHC and shared approximately 70% identity with mammalian PDIA3, and its mRNA was primarily expressed in the liver. PDIA3 was significantly increased in the liver of Atlantic salmon exposed to hyperoxic water during smoltification. Also Mn superoxide dismutase (Mn-SOD) and CCAAT/enhancer binding protein (C/EBP), other markers of oxidative stress, were upregulated by hyperoxia. Furthermore, PFOS exposure of hepatocytes resulted in elevated mRNA expression of PDIA3, Mn-SOD and C/EBPδ as well as peroxisome proliferator-activated receptor gamma (PPARγ). These results indicate a signaling connection between oxidative stress and ER stress. PDIA3 and C/EBPδ may be valuable markers in fish for exposure and effect to environmental stress.     

Alkaline preserved herring by-products in feed for Atlantic salmon (Salmo salar L.)

The aim of the present study was to investigate the digestibility, growth and slaughter quality in Atlantic salmon (Salmo salar L.) fed a novel type of moist feed. The moist feed was prepared from alkaline preserved (pH 11.2) herring (Clupea harengus) filleting by-products, mixed with a crude binder based on seaweed before the feed was shaped in a pelleting process and the final structure was set immersing the pellets in an acidic bath. The feeding experiment was carried out with seawater-adapted salmon with an average initial weight of 1.7 kg. The moist feed and a control feed were fed to three groups of salmon with 190 fish per group. The coefficient of total tract apparent digestibility of fat, protein and energy in fish fed the moist feed was 0.96, 0.81 and 0.87, respectively. The weight gain for fish fed moist feed was significantly higher compared to salmon fed commercially extruded control feed. Carcass quality data showed that fish fed the moist feed had significantly higher dressing out percentage and less visceral fat compared to fish fed the control feed. It is concluded that alkaline preserved fish by-products can be efficiently utilized in a novel moist feed technology, yielding good growth and digestibility in Atlantic salmon.     

Ghrelin receptor (GHS-R)-like receptor and its genomic organisation in rainbow trout, Oncorhynchus mykiss

Ghrelin, a GH-releasing and appetite-regulating peptide that is released from the stomach is an endogenous ligand for growth hormone secretagogue-receptor (GHS-R). Two types of GHS-R are accepted to be present, a functional GHS-R1a and GHS-R1b with unknown function. In this study, we identified cDNA that encodes protein with close sequence similarity to GHS-R and exon–intron organization of the GHS-R genes in rainbow trout, Oncorhynchus mykiss. Two variants of GHS-R1a proteins with 387-amino acids, namely DQTA/LN-type and ERAT/IS-type, were identified. In 3'-RACE PCR and genomic PCR, we also identified three GHS-R1b orthologs that are consisted of 297- or 300-amino acids with different amino acid sequence at the C-terminus, in addition to the DQTA/LN-type and ERAT/IS-type variations. Genomic PCR revealed that the genes are composed of two exons separated by an intron, and that two GHS-R1a and three GHS-R1b variants are generated by three distinct genes. GHS-R1a and GHSR-1b mRNA were predominantly expressed in the pituitary, followed by the brain. Identified DQTA/LN-type or ERAT/IS-type GHS-R1a cDNA was transfected into mammalian cells, and intracellular calcium ion mobilization assay was carried out. However, we did not find any response to rat ghrelin and a homologous ligand, des-VRQ trout ghrelin, of either receptor in vitro. We found that unexpected mRNA splicing had occurred in the transfected cells, suggesting that the full-length, functional receptor protein might not be generated in the cells. Gene structure and characterization of protein sequence identified in this study were closely similar to other GHS-R, but to conclude that it is a GHS-R for rainbow trout, further study is required to confirm activation of GHS-R1a by ghrelin or GHS. Thus we designated the identified receptor proteins in this study as GHS-R-like receptor (GHSR-LR).     

An analysis of spatial and environmental factors influencing hybridization between native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss) in the upper Kootenay River drainage, British Columbia

Westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) and introduced rainbow trout (O. mykiss, RBT) readily hybridize and introgression has occurred in many drainages across the historic native range of WCT. In British Columbia (Canada), the upper Kootenay River drainage is the heart of the westslope cutthroat trout (WCT, Oncorhynchus clarki lewisi) distribution in Canada this drainage harbours native WCT gene pools that are thought to be under threat from hybridization with introduced rainbow trout (RBT, O. mykiss). In this study, we assess the extent and distribution of WCT × RBT hybridization in the upper Kootenay River drainage. We used four diagnostic nuclear loci to determine the extent of hybridization in 981 fish collected from 23 sample localities across 12 different streams in the upper Kootenay River drainage. About 14% (142/981) of individuals were identified as hybrids (an individual with both RBT and WCT alleles), 3.4% (33/981) were identified as pure RBT, and the remaining individuals were identified as pure WCT. Although pure RBT were absent from the majority of locales (20/23), we found evidence of hybridization at 78% (18/23) of the localities and the percentage of heterospecific alleles (% I) ranged from 0.7% to 97.1%. Only 22% (5/23) of the localities showed no evidence of hybridization. Spatial analysis showed clustering among hybridized locations and decreasing hybridization with increasing distance from Koocanusa Reservoir, suggesting that the reservoir acts as a RBT source. We found no evidence that stream order, stream magnitude, or stream elevation influenced the extent of hybridization among localities. We compared our results to an analysis conducted in 1986, which indicated that hybridization is relatively recent in the upper Kootenay River drainage and that it is increasing in magnitude and distribution. In the absence of timely management intervention, the genetic integrity of WCT populations in the heart of their Canadian range may be lost. Our results indicate the dynamic nature of hybridization in fluvial systems and that for closely related taxa such as WCT and RBT, hybridization appears to be largely influenced by physical barriers to dispersal and contact between species.     

Dietary carotenoid pigment supplementation influences hepatic lipid and mucopolysaccharide levels in rainbow trout (Oncorhynchus mykiss)

We assessed the effects of dietary carotenoid pigment supplementation on liver histochemistry in the rainbow trout. One hundred and eight rainbow trout (mean mass 266 ± 10 g) were assigned to each of three replicate tanks for each of three dietary treatments; astaxanthin, canthaxanthin, or control at a target dietary inclusion of 100 mg/kg, by top-coating a pigment-free commercially extruded basal diet (Trouw Aquaculture, U.K.). Fish were fed for 3 weeks at a ration of 1.2% body mass/day, in a recirculating freshwater system maintained at 16 °C. Frozen liver sections were stained for total lipids, unsaturated lipids, glycogen, mucopolysaccharides, glycogen phosphorylase and aspartate aminotransferase. Relative amounts were measured quantitatively by image analysis. Carotenoid treatment significantly (P < 0.05) altered the total lipid profile and hepatic mucopolysaccharide contents of livers of rainbow trout. Results are discussed in relation to the catabolic potential of the liver in carotenoid pigment metabolism.     

Temporal variations in the diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri R.) in Rutland Water

The diet of brown trout (Salmo trutta L.) and rainbow trout (S. gairdneri Richardson) in Rutland Water were compared during the first two fishing seasons (April–October 1977 and 1978).Fortnightly samples of approximately forty stomachs were obtained from boat and bank, rod-and-line caught trout giving a total of 1046 stomachs over the two seasons.During 1977 seasonal changes in the diet were divided into two phases; the first being a period of abundant drowned terrestrial food until June. This was followed by a period of more stable water level from July onwards when chironomid larvae and pupae were consistently the most important food items and the diversity of food also increased.In 1978 the proportion of chironomid pupae and larvae declined and they were replaced in the diet by Gammarus and Asellus.     

Identification of glycoproteins in goblet cells of epidermis and gill of plaice (Pleuronectes platessa L.), flounder (Platichthys flesus (L.)) and rainbow trout (Salmo gairdneri Richardson)

Synopsis A quantitative analysis has been made of the glycoproteins present in the goblet cells of the epidermis, gill filaments and gill lamellae of three species of teleost fish. The glycoproteins have been identified by a combination of techniques, including the use of the enzyme sialidase followed by Alcian Blue staining, at pH 2.6 or I. o, in combination with periodic acid-Schiff. The selected fish were representative of species living in marine, freshwater and estuarine environments.The range of glycoproteins identified in these fish was similar to that found in mammalian tissue in that both neutral and acid glycoproteins were present, the latter included both sialomucins sensitive and resistant to sialidase, and sulphomucin. A single goblet cell contained either neutral or acid glycoproteins alone or in combination. Only the epidermis of the plaice and rainbow trout contained uniform cell populations producing acid glycoproteins, the former sulphomucin and the latter mainly sialomucin. At each site in the flounder and in the gill epithelia of the plaice and rainbow trout, the goblet cell population was mixed, with cells producing each type of glycoprotein. The number of goblet cells producing each type of glycoprotein varied at each tissue site.     

Gastro-intestinal transport of calcium and cadmium in fresh water and seawater acclimated trout (Oncorhynchus mykiss)

Transport of calcium (Ca) and cadmium (Cd) was examined along the gastro-intestinal tract (GIT) of freshwater and seawater Oncorhynchus mykiss irideus (FWT and SWTies respectively) using in vitro and in vivo experiments. Based on known physiological differences between FWT and SWT which aid in regulating ion levels and osmolarity, we hypothesized that SWT would have lower rates of Ca uptake. Also, we predicted that Cd rates would also be lower because Cd is known to share a common transport mechanism with Ca. Kinetics of Ca and Cd transport were determined using mucosal salines of varying concentrations [1, 10, 30, 60, and 100 (mmol L^− 1 for Ca, μmol L^− 1 for Cd)]. Linear and saturating relationships were found for Ca for FWT and SWT, but overall SWT had lower rates. Linear and/or saturating relationships were also found for Cd uptake, but rates varied little between fish types. Elevated Ca had no inhibitory effect on Cd transport, and Ca channel blockers nifedipine and verapamil had little effect on Ca or Cd uptake. However, lanthanum reduced Ca transport into some compartments. A 21 day in vivo feeding experiment was also performed where FWT and SWT were exposed to control diets or Cd-spiked diets (552 μg Cd g^− 1 food). Whole body Cd uptake between fish types was similar, but the majority of Cd in SWT remained in the posterior intestine tissue, while FWT transported more Cd through their gut wall. Overall it appears that large differences in Ca and Cd uptake between FWT and SWT exist, with SWT generally having lower rates.