Induction of metallothionein synthesis in rainbow trout,Salmo gairdneri,during long-term exposure to waterborne cadmium

Rainbow trout were exposed to 200 g cadmium/l in the water during four months at 6–10°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc, metallothionein and metallothionein mRNA. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The tissue zine and copper concentrations showed no major alterations during the experiment. The cytosolic distribution of cadmium, copper and zinc was followed during four months of exposure by Sephadex G-75 chromatography. It was found that cadmium was predominantly associated with proteins of an apparent molecular vieght of 10,000 daltons. These proteins were further identified as metallothioneins after fast protein liquid chromatography on a Mono-Q column. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after only one month. The kidneys reached significantly elevated levels of metallothionein in the exposed group after three months. In the gills, elevated metallothionein concentrations were observed after four months of exposure. After four months of exposure, the metallothionein mRNA content of liver and kidney was analyzed using a rainbow trout anti-sense RNA probe. Elevated MT mRNA levels were observed in both kidney and liver. These results demonstrate thatde novo synthesis of metallothionein is induced by cadmium in rainbow trout after exposure to the metalvia water.     

Cadmium detoxification effect of Chinese parsley Coriandrum sativum in liver and kidney of rainbow trout Oncorhynchus mykiss

Although fish feed supplemented with Chinese parsley (CP), Coriandrum sativum, does not affect the health of rainbow trout Oncorhynchus mykiss, it has the remarkable effect of reducing the uptake of Cd (12–17% compared with controls without CP). Here, we measured the change in the amount of metallothionein (MT) in the liver and kidney using gel permeation chromatography-high-performance liquid chromatography (TSK GEL SW 3000 column). The increase in MT content over time in the Cd-fed groups corresponded to the change in Cd accumulation. Gel permeation was performed with soluble fractions from the fish liver and kidney, and changes in the Cd, Cu, and Zn concentrations over time for each fraction, and the relationship between the form of Cd that accumulated in tissues and its toxicity were measured. A nontoxic MT fraction of 7,000 Da and a high-molecular-weight fraction of approximately 60,000 Da were isolated from the soluble fractions. The induction of nontoxic MT–Cd was higher in the CP-supplemented groups than in the control groups. The synergetic actions of several compounds in CP may contribute to bind MT, thereby preventing accumulation of harmful heavy metals and essential metals in fish.     

Effect of cadmium binding form on rabbit aminolevulinate dehydratase in high molecular weight fraction of rainbow trout liver

Enzyme inhibition owing to cadmium (Cd) accumulation in the soluble high molecular weight fraction (HMF) of the liver from rainbow trout was investigated by measuring the inhibition of aminolevulinate dehydratase (ALAD). Factors affecting ALAD, which reacts specifically to metal, were examined before measuring ALAD activity. Although enzyme inhibition by Cd in the HMF was confirmed in the excretion 2 (E2) group fed Chinese parsley (CP), it was weaker than that in the excretion 1 (E1, control) group, probably due to the chelation of Cd by some substances contained in CP. Based on the absorption spectrum, the proportion of Cd bound to cysteine thiol radicals in the HMF of the E2 group was greater than that of E1. The HMF might detoxify harmful metals to some extent because it contains relatively higher levels of cysteine residues than metallothionein (MT) and shows comparatively weak enzyme inhibition. The results of the present study demonstrated that, in addition to MT, HMF is involved in the detoxification of harmful heavy metals in fish.     

Metallothionein and metal levels in liver,gills and kidney of <Emphasis Type="Italic">Sparus aurata</Emphasis> exposed to sublethal doses of cadmium and copper

The levels of metallothionein (MT), a biomarker of metal exposure, and of Cd and Cu, known as MT inducers, were investigated in Sparus aurata intraperitoneally injected with 500 μg/kg of Cu and Cd for 2 days. MT and metal concentrations (Cd and Cu) were determined in liver, gills and kidney. MT levels were significantly increased in all investigated tissues, with the highest value in liver of Cu as Cd-treated fishes (3.56-fold and 3.3- fold, respectively). Metal concentrations were statistically different between all tissues. Highest metal concentrations were in the liver. The higher metal concentrations and MT induction levels support the main role of MT in metal homeostasis and detoxification.     

Cloning and expression of metallothionein cDNA in the hard clam (Meretrix lusoria) upon cadmium exposure

The objective of this study was to determine whether metallothionein (MT) expression was related to Cd accumulation in hard clams (Meretrix lusoria). We cloned clam MT. Then a specific primer for the hard clam was designed for MT mRNA measurements using real-time PCR. The hard clams were exposed to 50, 200, and 1000 μg/L cadmium (Cd) for 1, 3, 5, and 7 days, to determine the relationship between MT mRNA relative quantity (RQ) levels and Cd accumulation. The following results were obtained: (1) the cloned MT, consisting of 228 bp encoding a protein of 76 amino acids showed higher than 55% identity with the deduced amino acid sequence previously published for another bivalve. (2) Both MT mRNA expression and Cd accumulation appeared to be dose-responsive following cadmium treatment for 1, 3 and 5 days, respectively; however, no time-dependent responses were found. According to these results, MT mRNA expression levels can be used as an indicator to monitor the concentrations of Cd in aquaculture farms.     

Withdrawal times of freshwater rainbow trout,Salmo gairdneri Richardson,after treatment with oxolinic acid,oxytetracycline and trimetoprim

Abstract. Oxolinic acid (OA), oxytetracyline (OTC) and trimetoprim (TMP) were investigated in whole gutted rainbow trout, Salmo gairdneri Richardson, and in their skin, muscle and blood, at 6, 12 and 18°C. Concentrations of drugs were measured by high-pressure liquid chromatography. It is recommended that withdrawal times for rainbow trout be based on whole gutted rainbow trout and not, as in earlier investigations, from residual concentrations in muscle. Any concentrations in rainbow trout feed were found to be stable during the pelleting process used and up to 81 days after pelleting. The absorption of the drugs in rainbow trout show different temperature dependencies. A quantitative comparison of our overall data from this work with individual results from earlier investigations was undertaken. Suggested withdrawal times for freshwater rainbow trout treated with these drugs are: for OTC, 90 days below 6°C, 70 days between 6 and 12°C and 60 days above 12°C; for TMP, 60 days below 8°C and 40 days above 8°C; for OA, 20 days at approximately 18°C.     

Inter‐specific hybridization between Black Sea trout (Salmo labrax Pallas, 1814) and rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

Thermal shock‐induced triploid and unshocked control hybrids between rainbow trout (Oncorhynchus mykiss) and Black Sea trout (Salmo labrax) and their parental species were produced under hatchery condition by using heat shocks. Triploidization reduced eyed egg rate and alevin yield in all groups. Low survival rate was observed in both shock‐induced triploid hybrid and non‐shock‐induced control hybrids. Although hybrids demonstrated low body weight during the first feeding stage, they reached higher body weight by day 200 when compared with Black Sea trout and rainbow trout. A higher specific growth rate was calculated as 3.60 in the triploid hybrid groups, 1.41 in the triploid Black Sea trout groups and 2.27 in the triploid rainbow trout groups between days 110 and 200. A lower condition factor was determined in the hybrid than in the diploid parental species. A negative value of mid‐parent heterosis (MPH) performance was deected for condition factor, and a favourable MPH was detected for specific growth rate and weight in hybrids.     

Alterations in iron, zinc and major plasma proteins of rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), with sterile peritonitis induced by oil-adjuvanted multivalent bacterin vaccination

The early humoral responses of rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), with sterile inflammation induced by intraperitoneal Lipogen Triple vaccination were compared to determine if genetic differences in susceptibility to furunculosis in salmonids correlated with different acute phase responses to vaccination. Similar severe acute sterile peritonitis occurred in response to Lipogen Triple in both species. Both species also had a rapid transient reduction in plasma iron concentration at 3 days. Moderate hypoferraemia persisted to day 14 in brook trout, but returned to normal by day 7 in rainbow trout. Plasma zinc decreased sharply 3 days after vaccination in rainbow trout and returned nearly to control levels by day 10; however, plasma zinc did not change in brook trout. Two-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis of plasma proteins revealed that increased amounts of a 48-kDa protein group coincided with the hypoferraemic response in rainbow trout. In addition, a modest elevation in a 16-kDa protein group also occurred in rainbow trout. These studies demonstrated the rapid changes in plasma iron in both species and mild elevation of two putative acute phase plasma proteins associated with vaccine-induced inflammation in rainbow trout.     

文蛤金属硫蛋白基因的克隆与分析

金属硫蛋白是一种普遍存在于生物体内的低分子量、半胱氨酸含量丰富、易于被外界刺激诱导的金属结合蛋白。采用表达序列标签（EST）法,首次获得了文蛤金属硫蛋白（Mm—MT）的全长eDNA序列,序列全长657bp,开放阅读框长度为231bp,可以编码76个氨基酸。编码的氨基酸中半胱氨酸含量丰富,达27．6％,含有软体动物等无脊椎动物金属硫蛋白的特征序列。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员。     

Nitrogen utilisation and ureogenesis as affected by dietary nucleic acid in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima)

We evaluated the influence of dietary nucleic acid (NA) supplied either as an RNA extract (RNA) or as brewer's yeast (BY) on nitrogen (N) utilisation, N excretion and ureogenesis in a freshwater teleost, the rainbow trout (Oncorhynchus mykiss) and a marine teleost, the turbot (Psetta maxima). Five diets containing two levels of NA or BY were formulated for each species, and fed to juvenile rainbow trout (initial body weight (IBW): 21.7 g) and turbot (IBW: 11.8 g) over 8–10 weeks. Besides growth and N utilisation, we monitored total ammonia-N and urea-N excretion rates and measured the activities of selected enzymes (glutamate dehydrogenase, ornithine carbamoyltransferase, arginase and uricase). There was no clear N sparing effect of dietary NA or BY supplementations in either species. Feeding diets containing the RNA extract led to an almost two-fold increase in postprandial plasma urea-N and uric acid concentrations and in urea-N excretion rates. Glutamate dehydrogenase and arginase activities were decreased in fish fed NA supplemented diets. Uricase activities were inversely related to dietary NA levels. Data on N excretion and enzyme activities clearly suggest that ureogenesis is influenced by dietary NA in both species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Optimization of treatment regimes for controlled sex differentiation and sterilization in wild rainbow trout (Salmo gairdneri Richardson) by oral administration of 17α-methyltestosterone

The oral administration of 17α-methyltestosterone (MT) to wild rainbow trout fry at concentrations ranging from 1 to 100 mg/kg diet for periods up to 120 days following swim-up produced male-female ratios substantially different from controls. 100% males were produced with low MT doses (1, 3 and 9 mg/kg diet) administered for 60 days. High proportions (79–90%) of sterile rainbow trout were obtained with greater doses of the steroid (25, 50 and 100 mg/kg diet) fed for 120 days. A paradoxical feminizing effect at high doses of MT (50 and 100 mg/kg diet) was observed irrespective of the length of the treatment. No dose-related mortality during the treatments was observed.     

Passive immunization of rainbow trout,Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum,the causative agent of bacterial coldwater disease and rainbow trout fry syndrome

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS), causes high mortality in cultured salmonids. The present study was designed to determine the role antibody plays in conferring protection to rainbow trout fry, Oncorhynchus mykiss (Walbaum), by passive immunization with convalescent serum or serum from adult rainbow trout immunized with F. psychrophilum, and goat anti-F. psychrophilum serum. In each experiment, rainbow trout fry were injected intraperitoneally with antiserum and challenged by subcutaneous injection with a virulent strain (CSF-259-93) of F. psychrophilum 24-h post-immunization. Relative percentage survival (RPS) ranged from 9-42% when rainbow trout fry (mean weight 1.3 g) were injected with a 1:2 dilution of 25 microL of convalescent serum ranging in enzyme-linked immunosorbent assay antibody titres from 1600-102400. Rainbow trout fry (mean weight 1.0 g) passively immunized with 25 microL of serum from immunized adult fish exhibited RPS values of up to 57%. In each of these experiments, RPS increased with increasing antibody titres against F. psychrophilum. Passive immunization with 25 or 50 microL goat anti-F. psychrophilum serum, however, did not confer protection to fry (mean weight 1.3 g). These results suggest that trout antibody plays a role in conferring protection to F. psychrophilum, but antibody alone is unable to provide complete protection.     

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.     

The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.     

Evolutionary implications of two rainbow trout growth hormone genes

The primary structures of two rainbow trout growth hormone mRNAs (GH1 and GH2) have been deduced by direct sequencing of their respective cDNA clones and portions of the mRNA. Both GH1 and GH2 mRNA contain open reading frames comprised of 630 nucleotides and encode 210 amino acid residues of which 11 are variant. The translated regions of both mRNA are flanked by a short but rather conserved 5′-end, and a relatively long but highly diverged 3′-end. The differences at translated and 3′-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. The GH1 and GH2 mRNA are likely transcribed from two distinct loci which were duplicated during tetraploidization of salmonid genome between 50 to 100 million years ago. The GH2 gene has been isolated and sequenced from a rainbow trout genomic library. This gene spans a region of approximately 4 kilobases. The trout GH gene is comprised of 6 exons and 5 introns, in contrast to 5 exons and 4 introns in mammals. The additional intron in the trout gene interrupts the translated regions that are analogous to the last exon of the mammalian counterpart. The alleged internally repeating sequences in mammalian GH, prolactin (Pr1) and placental lactogen (PL) are not observed in the predicted polypeptide sequence of trout GH. In addition, direct repeats that flank exons I, III and V of mammalian GH, Pr1 and PL genes are absent in trout gene. These findings indicate that the rainbow trout GH gene structure does not support the current hypothesis that internally repeated regions in GH, Pr1 and PL arose from a small primordial gene.     

Elevated serum levels of alpha foetoprotein (AFP)-like immunoreactivity in rainbow trout, Oncorhynchus mykiss (Walbaum), with aflatoxin B1 induced hepatocellular carcinoma

Abstract. Alpha foetoprotein (AFP)-like immunoreactivity was detected by double immunodiffusion in thesera of adult rainbow trout, Oncorhyachus mykiss (Walbaum) with histologically confirmed hepatocellular carcinoma experimentally induced in microinjection of embryos with aflatoxin B₁ (AFB₁). Partial characterization indicated that this AFP-like immunoreactivity gave immunologic cross-reaction of partial identity with human AFP. had alpha₁ electrophorctic mobility, and an estimated molecular weight of 72kDa. Elevated serum AFP-like immunoreactivity levels were also measured by RIA in seven out of eight adult rainbow trout with hepatocellular carcinoma, and in three out of 17 AFB₁-exposed trout without demonstrable grosws or microscopic evidence for hepatocellular neoplasms. No serum AFP-like immunoreactivity was detected or measured in normal healthy age matched DMSO-control rainbow trout. These data are consistent with the following conelusions: (1) AFP-like immunoreactivity detected and measured in the sera of rainbow trout with hepaloecllular careinoma is an analogue to human AFP; and (2) the elevated serum AFP levels measured in adult rainbow trout wilh hepatocellular careinoma resrmble those found in humans with this same malignancy. These data further suggest that serum AFP measurements might be useful to confirm the appearance of hepatocellular carcinoma in experimental fish carcinogen-assay systems, and to detect hepatocellular neoplasia in high-risk wild fish populations exposed to carcinogenie pollutants.     

Protection of turbot, Scophthalmus maximus (L.), and rainbow trout, Oncorhynchus mykiss (Richardson), against vibriosis using two different vaccines

Abstact. The potency of a whole-cell bacterin (WCB) and a toxoid enriched whole-cell vaccine (WCEB) administered intraperitoncally into rainbow trout, Oncorhynchus mykiss (Richardson), were compared. The most effective vaccine was further evaluated by bathing turbot, Scophthalmus maximus (L.). These vaccines were composed of three strains of V. anguillarun , of the serotypes 01 and 02. Both vaccines conferred the highest protection against strains of serotype 01 within 4 weeks. With the toxoid enriched vaccine giving the best results (77 RPS). When trout were revaccinated after 7 weeks with this vaccine, good protection was achieved against strains of serotypes 01 and 02. Interestingly, when the WCEB was administered by bath to turbot, acceptable levels of protection against strains of both serotypes were obtained after 4 weeks of immunization.