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Influence of dietary ascorbic acid on plasma lipid levels in the rainbow trout.

Rainbow trout maintained on ascorbic acid deficient and three grades of ascorbic acid supplemented diets (160--1280 mg ascorbic acid pr kg feed) for a period of 53 weeks, were studied for effects on plasma lipid levels. Fish fed the diet with no ascorbic acid manifested lethargy, lordosis, scoliosis, internal hemorrhages and low body weight. With regard to blood properties, ascorbic acid deficient fish had low hematocrit and high plasma levels of triglycerides and cholesterol but low level of free fatty acids. Plasma level of free fatty acids was low also in fish fed the diet containing the highest amount of ascorbic acid.

Administration, Oral

Influence of thermal acclimation on membrane lipid composition of rainbow trout liver.

Rainbow trout (Salmo gairdneri) acclimated to 5 degrees C possessed larger livers and less neutral lipid per gram of liver than 20 degrees C-acclimated animals; quantities of liver glycolipid, phospholipid, and cholesterol did not vary significantly with acclimation temperature. The relative proportions of phosphatidylethanolamine increased significantly following cold exposure, whereas the quantities of sphingomyelin and cardiolipin declined. For all phosphatides examined (phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, lysolecithin, cardiolipin, sphingomyelin) cold acclimation resulted in 1) an increase in the quantity of polyunsaturated fatty acids, 2) a reduction in the level of saturated fatty acids, and 3) little change in the total content of monoenes and dienes. The increased content of polyunsaturated fatty acids in choline and ethanolamine phosphatides following cold acclimation was confined to the 2-position and occurred at the expense of monoenes and dienes. The relative proportions of n - 3 fatty acids, and less frequently n - 6 fatty acids, increased in phosphatides of cold-acclimated trout, whereas the relative proportions of n - 9 fatty acids declined. These data suggest a preferential incorporation of fatty acids belonging to the linolenic acid family at reduced temperatures. Temperature-induced changes in the chemical composition of trout liver phospholipids counteracted the effects of acute temperature change on nonelectrolyte permeability of isolated liposomes.

Adaptation, Physiological

A proposed nucleotide sequence for the 5S ribosomal ribonucleic acid of rainbow trout (Salmo gairdneri).

Rainbow trout cell cultures have been exposed to 32P-labelled inorganic phosphate and the labelled RNA has been isolated. The 5S ribosomal ribonucleic acid (5S rRNA) was purified by polyacrylamide gel electrophoresis, then digested with RNase T1 or pancreatic RNase. The products of complete digestion were separated and their sequences determined. These analyses have allowed a sequence to be proposed which differs in eight positions from that of mammalian 5S rRNAs.

Animals

Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout.

Acclimation of rainbow trout to 5, 15 and 22 degrees C for periods exceeding 4 months had no significant effect on the oxygen affinity of whole blood or on the concentration of ATP, which is the main organic phosphate in red cells. Slight differences were, however, found in the oxygenation properties of the haemolysates, which correlate with changes in the relative concentration of the multiple haemoglobins. The oxygen-binding properties of the main haemoglobin components account for the observed differences in the haemolysates. The possible thermoacclimatory significance of changes in haemoglobin multiplicity and co-factor concentrations is discussed.

Adaptation, Physiological

Effects of temperature, salinity, and feeding on aminotransferase activity in the liver and white muscle of rainbow trout (Salmo gairdneri Richardson).

1. The liver-somatic index of rainbow trout is governed by temperature and salinity, and by the interaction of these two factors. 2. The overall liver-alanine aminotransferase activity (in units/100 g body weight) increases slightly with increasing salinity of the surroundings in the case of rainbow trout. 3. The overall liver-aspartate aminotransferase activity (in units/100 g body weight) in rainbow trout depends on their food and the temperature at which they are kept. 4. Salinity adaptation leads to reductions in the specific alanine and aspartate aminotransferase activity in the liver of rainbow trout. 5. The specific alanine aminotransferase activity in the muscle of starving rainbow trout kept in diluted seawater (580 mOsm/l, 18 degrees C) is clearly higher than in control animals kept in tapwater.

Alanine Transaminase

Aflatoxin B1 induction of hepatocellular carcinoma in the embryos of rainbow trout (Salmo gairdneri).

Liver cancer in rainbow trout was induced by exposure of fertile eggs to an aqueous, 0.5 ppm (microgram/ml) solution of aflatoxin B1 (AFB1) for 1 hour. Single treatments, given on alternate days during the embryonic period, produced a low cancer incidence (less than 20%) prior to formation of the embryonic liver on day 14, but a steadily increasing incidence from day 15 (31.7%) until day 23 (58.3%), in fish examined 1 year later. Treatment of trout eggs with [14C]AFB1 was used to quantitate the amount of AFB1 absorbed by the eggs. Twenty-one-day-old rainbow trout eggs absorbed approximately 30 ng of [14C]AFB1 during a 1-hour exposure to 0.5 ppm aqueous [14C]AFB1. After 1 day 85-90% of the [14C]AFB1 was either metabolized and excreted or leached from the egg. The residual [14C]AFB1 remained constant until hatching when an additional 50% was lost. Comparison of the amount of AFB1 absorbed by eggs with the amount of AFB1 consumed per fish during a 1-year feeding trial at 4 ppb in the diet indicates that the trout embryo is even more sensitive than juvenile trout to the carcinogenic properties of AFB1.

Aflatoxins

Identification and isolation of protamine messenger ribonucleoprotein particles from rainbow trout testis.

Treatment of rainbow trout testis polyribosomes with ethylenediaminetetraacetic acid released polyadenylated protamine messenger RNA in the form of a ribonucleoprotein (mRNP) particle. This mRNP particle which sedimented at 12-14 S could be identified by hybridization to [3H]poly(U) and was partially purified by two successive sucrose gradient sedimentations. When RNA was extracted from the mRNP particle and used as a template in the wheat germ cell-free protein synthesizing system the sole product of translation was protamine. Of this RNA, 30% contained poly(A) sequences and was shown to comigrate with polyadenylated protamine messenger RNA during polyacrylamide gel electrophoresis. When the proteins of the mRNP particle were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two prominent polypeptides with apparent molecular weights of 73 000 and 29 000 appeared reproducibly. Treatment of trout testis polyribosomes with puromycin in the presence of 0.5 M KCl released a smaller (8-10S) mRNP particle which was similarly shown to contain protamine messenger RNA. Trout testis postribosomal supernatant fraction possessed 16-18S mRNP particles containing polyadenylated RNA which cosedimented with protamine messenger RNA when the particles were dissociated with sodium dodecyl sulfate.

Animals

Carcinogenicity and acute toxicity of dimethylnitrosamine in rainbow trout (Salmo gairdneri).

Four-week-old rainbow trout (Salmo gairdneri) were fed diets containing 0, 3, 50, 200, 400, and 800 ppm dimethylnitrosamine (DMN) for 52 weeks. At the end of 52 weeks, the fish were fed a control diet without DMN for an additional 26 weeks. Samples were taken at 26, 52, and 78 weeks to determine tumor incidence. A dose-related carcinogenic response was established from these results, and an equation was derived to relate the level of the carcinogen to the hepatocellular carcinoma incidence. From a published dose-response study that used outbred Porton rats, a second equation was derived for comparison. Rats and trout were approximately equal in their sensitivity to DMN carcinogenesis. The median lethal dose after ip injection of DMN was 1,770 mg/kg body weight in rainbow trout. Relative to the range of 15-50 mg/kg body weight reported for several mammalian species, trout were resistant to the acute toxicity of DMN.

Animals

Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver.

Aflatoxicol, R0, was isolated from Mt. Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1. Its identity was confirmed by mass, infrared, and ultraviolet spectrometry. The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1. Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2. In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B. subtilis. Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated. The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1. The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites. The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium. The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma.

Aflatoxins

Evolution of the lymphoid system. II. Evidence for immunoglobulin determinants on all rainbow trout lymphocytes and demonstration of mixed leukocyte reaction.

Indirect fluorescent antibody analyses utilizing antiserum specific for rainbow trout serum immunoglobulin (Ig) demonstrated that essentially all rainbow trout lymphocytes from thymus, spleen, anterior kidney and peripheral blood possess either Ig determinants or structures which cross-react with those determinants found on serum Ig. Peripheral blood leukocytes from rainbow trout were found to be caple of participating in a mixed leukocyte reaction. This observation is discussed in terms of the evolution of T cell function.

Animals

Phenotypic and phylogenomic characterization of Lactococcus garvieae isolates from rainbow trout (Oncorhynchus mykiss) in Türkiye.

Lactococcosis is an important bacterial disease of farmed fish and causes substantial economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture. In this study, Lactococcus garvieae isolates recovered from rainbow trout farms in Türkiye were characterized using phenotypic, molecular, and phylogenomic methods. Among 32 presumptive Lactococcus isolates recovered from 127 dead rainbow trout, four were confirmed as L. garvieae and exhibited identical biochemical characteristics, Pulsed Field Gel Electrophoresis (PFGE) profiles, and broad growth tolerance across different pH, salinity, and temperature conditions. All isolates were presumptively classified as resistant to ciprofloxacin and florfenicol, while remaining susceptible to tetracycline and penicillin. Based on the AMR profiles, strain LG2, which exhibited the most susceptible antimicrobial profile among the isolates, was selected for whole-genome sequencing (WGS). WGS of the representative isolate LG2 generated a single 2,214,687-bp chromosomal contig with 38.5% GC content and 99.0% BUSCO completeness. In silico PCR assigned LG2 to serotype I, and the genome contained an intact capsule-associated cps/kps locus. The chromosomal lsa(D) determinant and an mdt(A)-like efflux-associated gene were detected, whereas no plasmid replicons or acquired quinolone or florfenicol resistance genes were identified, indicating discordance between the phenotypic and genomic AMR results. Taxonomic verification of 236 publicly available Lactococcus assemblies yielded 41 verified public L. garvieae genomes, which, together with LG2, formed a 42-genome within-species dataset. LG2 was most closely related to the Turkish isolate OS-37, sharing 99.96% ANI and differing by three core SNPs; both belonged to ST109, whereas the other Turkish isolates belonged to ST139. cgMLST identified a conserved genomic backbone, while pan-genome analysis identified 5,655 gene clusters and an open pan-genome characterized by a large cloud-gene fraction. These findings demonstrate the importance of species verification in Lactococcus population genomics and reveal substantial accessory-genome diversity within L. garvieae. The genomic features of LG2 provide a basis for future pathogenicity and immunogenicity studies, although experimental validation is required. Overall, these findings highlight the importance of local genomic surveillance for understanding L. garvieae population structure and provide a genomic framework for future region-specific vaccine research.

Animals

Distribution and elimination of naphthalene and 2-methylnaphthalene in rainbow trout during short- and long-term exposures.

The accumulation and elimination of 14C in rainbow trout tissues following short- and long-term exposures to aqueous 14C-naphthalene or 14C-2-methylnaphthalene were studied. After exposure for eight hr to 0.005 mg/L or 0.023 mg/L of 14C-naphthalene most tissues of fingerling rainbow trout studied contained 14C at 20 to 100 times the water levels while fat and bile contained 14C at several hundred times water levels. The half-lives for elimination of 14C from all tissues except fat were less than 24 hr. Exposure of fingerling rainbow trout to 14C-naphthalene or 14C-2-methylnaphthalene for four weeks in a continuous-flow delivery system resulted in maximum tissue levels of these chemicals of from 40 to 300 times the water concentration. Maximum bile 14C levels were 13,000 and 23,500 times the water concentration for 14C-naphthalene and 14C-2-methylnaphthalene exposure, respectively. Elimination of 14C accumulated from 14C-naphthalene in this long-term exposure was much slower than after short-term exposures, while elimination of 14C accumulated from 14C-2-methylnaphthalene was biphasic. The presence of parent compounds and metabolites in acetone extracts of muscles was determined by TLC. The data suggest that the biphasic release of 14C from muscle of trout exposed to 14C-2-methylnaphthalene may be due to differential elimination of parent compound and metabolites.

Animals

Production of coronary arteriosclerosis with sex hormones and human chorionic gonadotropin (HCG) in juvenile steelhead and rainbow trout, Salmon gairdneri.

Coronary arteries were examined for significant changes in myointimal hyperplasia in one-year old juvenile steelhead or rainbow trout treated with estradiol, testosterone or human chorionic gonadotropin (HCG). In one experiment juvenile steelhead trout were injected weekly with 2 microgram/g HCG, 5 microgram/g estradiol cypionate or 5 microgram/g testosterone cypionate for 50 days. Control fish were those killed prior to treatment or injected with fish saline or cottonseed oil. In two experiments juvenile nonanadromous rainbow trout were treated with estrogen and testosterone with the same weekly doses as juvenile steelhead for 42 and 30 days, respectively. In the first experiment juvenile rainbow were injected weekly with 2 microgram/g HCG. Juvenile rainbow treated with estradiol, testosterone and HCG and juvenile steelhead treated with testosterone had larger coronary lesions than untreated or control fish. Coronary hyperplastic nodules resembled morphologically those observed previously in sexually mature steelhead and rainbow trout. These data support the hypothesis that reproductive hormones are causal factors in spontaneously occurring arteriosclerosis in spawning Salmo gairdneri.

Animals

Chemoproteomics Prioritizes Mitochondrial ADP/ATP Translocase as a Candidate Target Associated with 6PPDQ-Induced Respiratory Toxicity in Rainbow Trout Gill.

6PPD quinone (6PPDQ) is an emerging contaminant that induces acute respiratory toxicity in rainbow trout (Oncorhynchus mykiss), yet its underlying molecular mechanisms remain poorly understood. In the present study, short-term in vivo exposure of rainbow trout to 6PPDQ resulted in substantial accumulation and limited biotransformation of 6PPDQ in the gill, accompanied by pronounced gill structural damage and increased whole-fish oxygen consumption. Taking advantage of the electrophilic reactivity of the quinone moiety of 6PPDQ toward cysteine residues, we applied activity-based protein profiling (ABPP) to gill tissue. ABPP revealed marked alterations in mitochondrial cysteine reactivity and highlighted ADP/ATP translocase (ANT) as a candidate 6PPDQ-interacting mitochondrial protein. A Cys-160-containing ANT peptide within the nucleotide-binding domain of ANT was pinpointed as the covalent binding site through ABPP, Peptide-centric Local Stability Assay (PELSA), and molecular docking. Functional assays using isolated gill mitochondria showed that 6PPDQ elicited an uncoupling-like mitochondrial respiratory response that was partially attenuated by the ANT inhibitor carboxyatractyloside (CATR), supporting the functional involvement of ANT in this gill-based model. Together, these findings nominate ANT as a candidate gill mitochondrial target associated with 6PPDQ-induced acute respiratory toxicity and demonstrate the utility of chemoproteomics for prioritizing mechanistically relevant protein interactions of emerging pollutants.

Animals

Serotypic and Genomic Diversity of Vibrio anguillarum in Rainbow Trout Farms in Turkey: Implications for Vibriosis Control and Vaccine Candidate Selection.

Outbreaks of vibriosis caused by Vibrio anguillarum are a persistent constraint on rainbow trout (Oncorhynchus mykiss) aquaculture. However, information on the population structure of field strains in Turkey has been lacking. Here, we report the first systematic serotypic, proteomic, and genomic characterization of 23 V. anguillarum isolates collected over 10&#x2009;years from rainbow trout farms located in six major aquaculture regions of Turkey. Serological analyses based on microagglutination, supported by ELISA characterization of hyperimmune sera, identified a clear predominance of serotype O1, whereas isolate V12 exhibited a non-agglutinating, atypical O-antigen profile. Protein profiling (SDS-PAGE) and immunoblotting showed largely conserved whole-cell protein patterns among the isolates, but distinct immunogenic bands at 14, 18, and 40&#x2009;kDa were detected in isolates V18 and V21. Long-read whole-genome sequencing revealed that most Turkish isolates grouped within the global O1 clade, while V12, V25, and V28 isolates occupied more distant branches. Comparative genomics demonstrated a conserved core virulence gene set (RTX toxins, siderophore and iron-uptake systems, motility and adhesion factors, Type VI secretion system), with strain-dependent variation in accessory loci such as anguibactin and T6SS-I. Experimental infections of rainbow trout demonstrated significant differences in virulence among isolates (p&#x2009;<&#x2009;0.05), with the V18 isolate showing high, the V15 intermediate, and the V12 low-mortality rates. By elucidating the relationship among the serotype, immunogenic protein profiles, virulence gene repertoires, and in&#xa0;vivo pathogenicity, this study provides a comprehensive overview of the antigenic and genomic diversity of Vibrio anguillarum isolates from Turkey. Notably, the identification of V18 and V21 as promising candidate strains for further vaccine evaluation, characterized by high virulence and unique immunogenic features, provides a scientific foundation for the development of serotype-specific vaccination strategies to mitigate vibriosis-associated losses in aquaculture.

Animals

The effects of temperature and thermal acclimation upon the osmotic properties and nonelectrolyte permeability of liver and gill mitochondria from rainbow trout (Salmo gairdneri).

Thermal acclimation of rainbow trout (Salmo gairdneri) taken from 20 degrees C to 7 degrees C resulted in adaptation of mitochondrial function, as evidenced by increases in the specific activities of NADH- and succinate-cytochrome c reductase of 1.93- and 2.7-fold respectively. Mitochondria from both gill and liver obeyed the Boyle-van't Hoff relationship in the range from 400 to 60 mosM. Thermal acclimation had no effect on the osmotic properties of liver mitochondria, whereas gill mitochondria from cold-acclimated trout were more sensitive to osmotic swelling than mitochondria from warm-acclimated individuals. The non-electrolyte permeability of liver mitochondria was assessed by optically monitoring mitochondrial swelling rates in isosmotic solutions of urea, glycerol, mannitol and glucose. Two parameters of mitochondrial swelling were determined: (a) initial swelling rates, d(1/A)dt, and (b) swelling constants, ks, derived from the time required to swell a fixed volume. Regardless of the assay temperature or the permeant employed, liver mitochondria from cold-acclimated trout exhibited greater initial swelling rates than mitochondria from warm-acclimated trout, indicating properties of temperature-compensated permeability. The apparent ranking of nonelectrolyte permeabilities was urea greater than glycerol greater than mannitol greater than glucose. ks values for urea and glycerol from cold-acclimated trout were greater than values typical of warm-acclimated populations; however ks values for glucose and mannitol were not influenced by thermal acclimation. Regardless of the permeant considered, activation energies for ks values were 3- to 5-fold greater than those for initial swelling rates. The time course of mitochondrial swelling consists of two components, an initial rapid swelling phase characterized by a half-life of 3-12 seconds, and a slower swelling phase characterized by a half life of 1-6 minutes. Initial swelling rates, which approximate the rapid swelling component, are considered to be the least ambiguous index of permeability, whereas ks values are more complex and strongly influenced by the slower swelling component.

Acclimatization

Microtubules and extracellular microtubule-like structures in the retina of the rainbow trout.

In the retina of the rainbow trout (Salmo gairdneri, Richardson) two types of microtubular structures are demonstrated. Besides the normal type of microtubules (about 200 A in diameter), occurring in all cell types of the retina, a second type is described which is termed microtubule-like structure (MLS) because of its extracellular localization. These MLS have a diameter of about 250 A under the same preparative conditions in which the normal microtubules appear 180-200 A thick. The interspace between the tubules is smaller than between the microtubules. Specific MLS to membrane associations exist, which are analyzed by serial sectioning and tilting procedures. It is suggested that the MLS have their origin at small membranous extrusions of the plasmalemma. These extrusions could contain nucleation sites for the MLS-formation within the extracellular space. It remains unknown which cell type produces the MLS proteins and which factors are responsible for the aggregation of the subunits to intact MLS.

Animals

Distribution and biliary excretion products of di-2-ethylhexyl phthalate in rainbow trout.

After 24-hr exposures of rainbow trout to 0.5 ppm of di-2-ethylhexyl [14C]phthalate, one-half of the radioactivity present in the fish was localized in the bile. The bile was pooled and fractionated by selective solvent extraction before and after beta-glucuronidase hydrolysis. Individual radioactive compounds were further separated and characterized by thin-layer chromatography. Gas chromatography-mass spectrometry was used to confirm the results of thin-layer chromatographic analysis. These procedures demonstrated that bile contained a number of DEHP metabolites, but only about 1% of unchanged DEHP. The major metabolite, mono-2-ethylhexyl phthalate glucuronide accounted for 72% of the total bile radioactivity. The remaining bile radioactivity was found to be present as phthalic acid glucuronide, mono-2-ethylhexyl phthalate and two partially characterized polar metabolites.

Animals