Antioxidative role of selenium against the toxic effect of heavy metals (Cd<Superscript>+2</Superscript>, Cr<Superscript>+3</Superscript>) on liver of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> Walbaum 1792)

The main purpose of this study is to discuss the effect of Cd⁺², Cr⁺³ and Se metals on biochemical parameters in liver tissue of Oncorhynchus mykiss. The rainbow trout were exposed to heavy metal stress (Cd⁺², Cr⁺³) at 2 ppm dosage. The present study was undertaken to determine the protective effect of selenium treatment at the same dosage (2 ppm) on some biochemical parameters. The activity of catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and the changes in levels of malondialdehyde (MDA) from biochemical parameters were determined in liver tissue of the fish groups exposed to heavy metals, especially for the selenium-applied groups. Results of this study showed that the activities of CAT, GSH-Px and SOD in the tissues of fish exposed to the stress of Cd⁺² and Cr⁺³ were significantly lower than the control groups (P < 0.05). Meanwhile, the closer values to the control groups were obtained in selenium-added groups (Cr⁺³ + Se⁺⁴, Cd⁺² + Se⁺⁴). For the level of MDA, the last production of lipid peroxidation showed increases (P < 0.05) in the groups exposed to the metal stress, whereas significant decreases were obtained in selenium-applied groups. The result of the statistical evaluation showed that the negative effects occurring in the biochemical parameters of the applied groups exposed to the toxicity of heavy metal were significantly eliminated (P < 0.05) as a result of selenium treatment.     

Stabilizing effect of Ca<Superscript>2+</Superscript> on myosin and myofibrils of squid mantle muscle as affected by heating conditions

The suppressive effects of Ca²⁺ on the thermal denaturation of myosin and myofibrils of squid mantle muscle were compared. The stabilization effect on myosin was smaller than that on myofibrils and was not affected by KCl concentration. The stabilizing effect of Ca²⁺ on myosin decreased as the heating temperature dropped, showing no stabilization at 20°C, while the effect on myofibrils was the same at all temperatures tested. The stabilizing effect of Ca²⁺ on myosin disappeared even at 30°C in the presence of sorbitol, where a small inactivation rate was found, while the effect of Ca²⁺ on myofibrils was equally detected irrespective of the reduction in inactivation rate in the presence of sorbitol. Stabilization of myosin by Ca²⁺ again appeared even in the presence of sorbitol when the heating temperature was raised to 38°C. It was suggested that Ca²⁺ confers stabilization on myosin only when myosin is under unstable conditions. The stabilization effect of Ca²⁺ on myosin was enhanced upon F-actin binding: Ca²⁺-bound myosin was more significantly stabilized by F-actin binding, and the effect was no longer affected by the conditions for heating.     

Antioxidative role of cerium against the toxicity of lead in the liver of silver crucian carp

The antioxidative role of cerium was investigated in the liver of silver crucian carp injected with lead. The fish were intraperitoneally injected with 10, 20, or 30 mg/kg wet weight PbCl₂. After a 14-day period of incubation, 35 animals were injected with a solution of 1.5 mg/kg wet weight CeCl₃. After 42 days, the wet weight and the liver weight of the fish were weighed, and the oxidative stress of the fish liver was estimated by assaying lipid peroxide, superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, glutathione, ascorbic acid, and reactive oxygen species (ROS). The results show that Ce³⁺ could decrease ROS accumulation, relieve the inhibition of the activities of the antioxidant enzyme and the reduction of antioxidants in fish liver caused by Pb²⁺, and decrease the enhancement of hepatosomatic index of fish under various Pb²⁺ dosages.     

Responses of antioxidant enzymes,lipid peroxidation,and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase in liver of the fish <Emphasis Type="Italic">Goodea atripinnis</Emphasis> exposed to Lake Yuriria water

Lake Yuriria, located in the heavily populated and polluted Mexican Central Plateau, receives domestic sewage, industrial effluents, and municipal wastewaters that are still directly discharged without treatment into the tributaries and the lake. Pollutants in water and sediments include heavy metals, aromatic hydrocarbons, and organochlorine pesticides. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), as well as Na⁺/K⁺-adenosine triphosphatase (Na⁺/K⁺-ATPase) activity, and lipid peroxidation (LPO) were evaluated in the livers of the fish Goodea atripinnis after 96 h of exposure to water collected in March and June 2005 from three sites: Y (limnetic zone), L (Lerma tributary), and C (la Cinta tributary). Physical and chemical parameters of the lake water were also analyzed. Increases in CAT activity and LPO levels at all three sites were detected compared with control fish (P < 0.05), while GPx and SOD activities decreased significantly (P < 0.05). Na⁺/K⁺-ATPase activities were similar to the control in fish exposed to limnetic water from both March and June but were higher than control at the two tributary sites in March (P < 0.05); fish exposed to water from the Lerma tributary in June exhibited lower Na⁺/K⁺-ATPase than the control (P < 0.05). During March, CAT and Na⁺/K⁺-ATPase activities were increasing more than in June in Y and L, respectively, while in June, SOD and GPx were depleted more than March in L and Y and L, respectively. Despite the antioxidant defenses of the fish liver, exposure to all water samples from Lake Yuriria exerted alterations in hepatic LPO levels, antioxidant enzymes, and Na⁺/K⁺-ATPase activities that could substantially impair the mechanisms of fish defenses against oxidative stress.     

Insight into nucleotide and Ca<Superscript>2+</Superscript> binding to carp α-actin

ABSTRACT:   Nucleotides and Ca²⁺ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca²⁺ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 10⁴/M and 1.2 × 10⁵/M, respectively. Competitive binding of Ca²⁺ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca²⁺ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.     

Anti-oxidative functions of <Emphasis Type="Italic">mt2</Emphasis> and <Emphasis Type="Italic">smtB</Emphasis> mRNA expression in the gills and brain of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) upon cadmium exposure

There were not any past studies about metallothionein isoforms (smtB and mt2) having anti-oxidative functions on zebrafish after Cd²⁺ exposure. On the other hand, the anti-oxidative enzymatic factors such as superoxide dismutase (sod), glutathione peroxidase (gpx1a), and catalase (cat) are used as references to investigate whether the smtB and mt2 have anti-oxidative responses on the gills and brain of zebrafish after 1–6 h of 0 and 1.78 μM Cd²⁺ exposure. The anti-oxidative system such as sod, cat, and gpx1a mRNA expressions demonstrated a cascade response upon Cd²⁺-induced oxidative stress in the present study. Interestingly, the smtB mRNA expression levels increased by 3.2- to 6.1-fold, and mt2 raised by 4.1- to 11.3-fold in gills at 1 and 3 h after exposure to Cd²⁺, respectively. On the other hand, the smtB mRNA levels increased by 10.6- to 58.6-fold, but mt2 mRNA levels increased by 2.3- to 11.1-fold in brain at 1 and 3 h after exposure to Cd²⁺, respectively. In addition, both tissues showed increased apoptosis levels at 3 h, and recovery after 6 h of Cd²⁺ exposure. From the results, we suggest that both mt2 and smtB play a role in anti-oxidation responses within 6 h after exposure to Cd²⁺. In conclusion, the smtB mRNA levels have a higher response than mt2 in the brain, but both mRNA expressions appear to have a similar pattern in the gill. We suggest that smtB plays an important role to defend oxidative stress in the brain of adult zebrafish upon acute Cd²⁺ exposure.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

The toxic effects of Cd²⁺ on Ca²⁺ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l^-1 of Cd²⁺ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca²⁺ uptake within 4h resulting in a great increase in K_m values for Ca²⁺ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca²⁺ influx of larvae in solutions of 0.2 mM Ca²⁺ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd²⁺ than 0 day-old larvae. Although the Ca²⁺ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l^-1 Cd²⁺ solution, total body Ca²⁺ content was significantly reduced in 3 day-old larvae. Increased Ca²⁺ uptake efficiency ensures sufficient Ca²⁺ for normal growth. However, rapid increase in Ca²⁺ influx after hatching also leads to higher Cd²⁺ uptake. Exposure to Cd²⁺ will lead to a drop in body Ca²⁺ content resulting in retardation of larval growth. Therefore, we conclude that if Ca²⁺ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca²⁺ and will show signs of Cd²⁺ poisoning.     

银离子对鱼类及其他水生动物的毒性

以白鲢(鱼种),罗非鱼(鱼种)、鲤(鱼种)、鲫(成鱼)、中华绒螯蟹(幼体)、克氏螯虾(成体)、蝌蚪等水生动物为材料,做银的毒性试验,同时分析含银水体中,鱼体内的银富集状况。试验结果表明:(1)银离子对这几种水生动物幼体的半致死浓度(LC_(50))仅在2—40ppb之间;但对成鱼的 LC_(50)要比幼体大得多。(2)在含银水体中,底层鱼类(如鲫)的银富集量最大的部位在内脏;上层鱼类(如?条)富集量最大的部位是鳃。鱼类中肌肉的富集量最小,其次是骨。     

Effect of dietary graded levels of cottonseed meal and gossypol on growth performance,body composition and health aspects of allogynogenetic silver crucian carp,Carassius auratus gibelio♀ × Cyprinus carpio♂

A 6‐month trial was conducted to evaluate the effects of dietary cottonseed meal (CSM) and free gossypol (FG) on allogynogenetic silver crucian carp, Carassius auratus gibelio♀ × Cyprinus carpio♂ with 4 replicates of each treatment. Isonitrogenous and isocaloric diets were formulated with the 0 g kg^?1 (control), 200 g kg^?1, 400 g kg^?1, and 560 g kg^?1 CSM. Diets with FG were made by supplementing batches of control diet with 214 mg kg^?1, 428 mg kg^?1, and 642 mg kg^?1. Weight gain, specific growth rate, and protein efficiency ratio increased significantly up to an inclusion level of CSM of 400 g kg^?1 in the diet, with a significant decrease in food conversion ratio. Further increase in CSM to 560 g kg^?1 did not cause further changes in fish performance. Free gossypol did not affect fish performance significantly at any inclusion level. Neither CSM nor FG caused significant effects in any of the other evaluated parameters such as whole body composition, haemoglobin concentration, activities of serum lysozyme, superoxide dismutase, alanine aminotransferase and aspartate aminotransferase, and histology of hepatic tissues and midgut. Our results suggested that crucian carp can tolerate at least 642 mg kg^?1 FG and that it is safe to including 400 g kg^?1 CSM in crucian carp feed.     

Effects of a dietary administration of purple coneflower (Echinacea purpurea) on growth,antioxidant activities and 8 miRNAs expressions in crucian carp (Carassius auratus)

Echinacea purpurea (EP), a globally popular herbal medicine, has been used to treat various diseases in human and animals. However, little has been reported about its effects in fish. In this study, crucian carp (Carassius auratus) were selected to evaluate the effects of EP on growth performance and antioxidant response and the expressions of microRNAs. The results showed EP could stimulate the growth of crucian carp with the best effect was observed at dose of 4 g kg^?1. In serum, the content of hydroxyl radical (·OH) and malondialdehyde (MDA) decreased by EP supplementation, whereas the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) increased. Similarly, the content of ·OH decreased, and the activities of CAT, GPX and GR increased in liver of crucian carp. Furthermore, in livers of crucian carp, EP supplements upregulated the expressions of the microRNAs (miR‐16, miR‐21, miR‐125b, miR‐146a, miR‐155, miR‐181a and miR‐223), which had been confirmed to participate in regulating antioxidant and immune function in mammals. Our results suggest EP supplements in diets stimulated growth performance and antioxidant response of crucian carp. In liver, the upregulation of specific miRNAs may participate in the antioxidant effects of EP diets.     

A study of the damage of the intestinal mucosa barrier structure and function of <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The aim of this study is to explore the effect of Aeromonas hydrophila on the intestinal mucosal barrier structure and intestinal permeability in grass carp (Ctenopharyngodon idella). Histopathological examinations showed that A. hydrophila induced severe intestinal lesions, including inflammatory cell infiltration and intestinal villus fusion and swelling. Messenger RNA (mRNA) expression of the inflammatory cytokines TNF-α, IL-1β, IL-8, IL-10 and MyD88 was significantly increased after infection with A. hydrophila. The permeability of intestinal mucosa was determined using Evans blue (EB) and d-lactic acid. The results indicated that the levels of EB and serum d-lactic acid were significantly increased after infection with A. hydrophila (p < 0.05). Our results also indicated that the intestinal mucosal barrier injury induced by A. hydrophila infection was closely associated with the expression of the tight junction (TJ) protein zonula occludens-1 (ZO-1), occludin, claudin b and claudin c as well as the activity of Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase. Lower mRNA levels of occludin and lower Na⁺, K⁺-ATPase and Ca²⁺, Mg²⁺-ATPase activity in the intestines were observed after challenge. ZO-1 and claudin c were significantly increased 24 h after infection with A. hydrophila. The most interesting finding was that claudin b also significantly increased 24 h after challenge and then decreased to lower levels at 72, 120 and 168 h post-infection compared to the PBS-treated control group. The results demonstrated that grass carp infection with A. hydrophila induced intestinal inflammation and impaired the structure and function of the intestinal mucosal barrier.     

微卫星分离模式显示雄性三倍体鲫产生非整倍体精子

三倍体鲫(Carassius auratus)行天然雌核发育,其自然种群中却有较高比例的雄性个体,这些雄性个体的性腺发育正常,能产生有活力的精子。而且其精子的DNA含量约为体细胞的一半,显示三倍体鲫精子发生过程中可能经历了均等的减数分裂。流式细胞术虽然能够较准确地测定细胞群的平均DNA含量,但是却很难检测到单个精子中的个别染色体增减,要明确回答雄性三倍体鲫产生的精子是否为整倍体需要定量地检测单个精子的遗传组成。本研究用微卫星标记检测以雌性鲤(Cyprinus carpio)与雄性三倍体鲫为亲本构建的杂种家系的基因型,结果发现母本鲤的多态位点在子代中呈孟德尔分离,父本三倍体鲫具有三套鲫基因组,其等位基因在子代中呈随机分离。上述研究结果提示:三倍体鲫起源于二倍体鲫的同源加倍,而非二倍体鲫和鲤的种间杂交;三倍体鲫通过染色体的随机分离产生非整倍体的精子,其精子发生过程中没有均等的减数分裂。三倍体鲫行雌核发育生殖,却可能并非起源于种间杂交且群体中的雄性个体可育,因而是单性生殖鱼类中的一个特例。     

Effects of sulfide on K+ flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K⁺ influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na⁺, K⁺-ATPase was attained at 10 mol l^–1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K⁺ influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K⁺ influx increased, but was inhibited to about 15% by 30 mol l^–1 sulfide. In RBCs of crucian carp neither Na⁺, K⁺-ATPase nor ouabain-resistant K⁺ influx were affected by sulfide concentrations up to 850 mol l^–1. Differences in sulfide-sensitivity of K⁺ influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na⁺, K⁺-ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K⁺ influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species.     

Comparison of behavioral responses to a novel environment between three teleosts,bluegill <Emphasis Type="Italic">Lepomis macrochirus</Emphasis>, crucian carp <Emphasis Type="Italic">Carassius langsdorfii</Emphasis>, and goldfish <Emphasis Type="Italic">Carassius auratus</Emphasis>

To investigate the emotional reactivity of fish in a novel environment, the swimway test was developed. The swimway apparatus consists of a shaded start chamber and an open, illuminated swimway. Fish were first introduced and habituated to the start chamber. A door partitioning the start chamber from the swimway was then opened, and behavioral responses of the fish in the apparatus were measured. By using the swimway test, behavioral responses to a novel environment of bluegill Lepomis macrochirus, crucian carp Carassius langsdorfii, and goldfish Carassius auratus were quantified and compared. The emotional reactivities in blue gill were found to be the lowest and crucian carp the highest, indicating bluegill are relatively active or ‘bold’, and that the crucian carp are relatively passive or ‘shy’, in a novel environment. It is suggested that the swimway test is applicable to examning inter-species differences in relative emotional reactivity or boldness in a simplified novel situation.     

Characterization of a pancreatic DNase from pyloric caeca of atlantic cod (Gadus morhua L.)

An alkaline deoxyribonuclease (DNase) from cod pancreatic tissue has been characterized. The enzyme is a DNase I type endonuclease and hydrolyzes effectively both native and denatured DNA. Monomeric actin inhibits the enzyme reaction. The enzyme obeys Michaelis-Menten kinetics and the apparent K_m value for native linear duplex DNA is 33 µg/ml. The cod DNase opens supercoiled plasmid DNA, by introducing adjacent nicks in both strands, possibly separated by 5–10 nucleotides. DNA hydrolyzed by cod DNase functions as substrates both for DNA polymerase and ligase, and the nicks therefore contain 5-phosphoryl and 3-hydroxyl groups. Optimum concentrations of divalent cations are 5 mM Mg²⁺, 0.63 mM Mn²⁺ and 0.075 mM Ca²⁺. However, Ca²⁺ is apparently not essential for the enzymatic functions. The enzyme has a narrow temperature optimum at 42°C and is thermolabile above 50°C; however, Mn²⁺ shifts the optimum slightly to 45°C by causing increased temperature stability. The cod DNase reaction is inhibited by the DNA intercalating compounds actinomycin D and ethidium bromide. Histidine-modifying reagents such as tosyl phenylalanyl chloromethylketone and diethyl pyrocarbonate inhibit the enzyme activity, but the cod DNase is insensitive to disulfide-reducing agents.     

Genetic characterization of the progeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii

Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm.     

Isolation and expression analyses of the <Emphasis Type="Italic">Sox9a</Emphasis> gene in triploid crucian carp

To investigate the evolutional significance of Sox9 in fish, we isolated and characterized Sox9a cDNA and genomic clones in triploid crucian carp. The cDNA encoded a protein of 457 amino acids with an HMG box and showed more than 60% amino acid sequence identity with known vertebrate Sox9 proteins. Triploid crucian carp and vertebrate Sox9s showed similar gene structure, and two introns in the coding region were located at conserved positions. On the basis of the amino acid sequences, Sox9a can be categorized into the same subgroup of Sox-E proteins as Sox8, 9, and 10. Interestingly, the expression of triploid crucian carp Sox9a was predominantly observed not in the ovary but in the testis by Northern blot and RT-PCR analysis. The expression analysis of Sox9a suggested that it may seldom contribute to the formation of normal functions of spermatozoa, but it may play an important role in the development of testicular tubules. Besides the testicular expression, Sox9a was also shown to be expressed in many other tissues including the brain, kidney, and heart of triploid crucian carp, indicating that Sox9 may have unique functions in some specific tissues during development.