Effect of enrofloxacin and emodin on heat-shock proteins’ expression in hepatic cells of grass carp (Ctenopharyngodon idellus)

The aim of the present study was to compare the expression pattern of heat-shock protein 60 (HSP60), 70 (HSP70), and 90 (HSP90) mRNA in hepatic cells of grass carp exposed to enrofloxacin and emodin concentrations. The expression pattern of different genes encoding heat-shock proteins in hepatic cells of grass carp was exposed to graded levels of enrofloxacin (12.5, 25, 50, 100, and 200 μg/ml) or emodin (0.04, 0.2, 1, 5, and 25 μg/ml) for 24 h and were investigated by real-time quantitative PCR. When cells were exposed to up 50 μg/ml enrofloxacin, both HSP60 and HSP70 mRNA levels firstly were increased and thereafter significantly dropped at high concentrations (P < 0.05), while HSP90 decreased with increasing enrofloxacin concentration. Besides, HSP60 and HSP70 expressions were significantly inhibited in the high-concentration groups. In addition, the HSP90 mRNA levels in the treatment exposed to 100 and 200 μg/ml enrofloxacin were significantly lower than those of the control group (P < 0.05). Furthermore, when cells were exposed to graded levels of emodin, HSPs (HSP60, 70, and 90) mRNA levels significantly increased in the groups exposed to 5 and 25 μg/ml of emodin. The different expression pattern of HSPs implied that enrofloxacin could inhibit the expression levels of HSPs, while optimal level of emodin could trigger higher expression levels of HSPs in hepatic cell of grass carp to protect against further damage.     

Both TGF-β1 and Smad4 regulate type I collagen expression in the muscle of grass carp,Ctenopharyngodon idella

Fish Physiology and Biochemistry - Type I collagen is proven to make an important contribution to fish muscle quality. Our previous study has shown the Smad4-dependent regulation of type I collagen...     

Bioaccumulation of hexachlorocyclohexane,dichlorodiphenyltrichloroethane, and estradiol-17β in catfish and carp during the pre-monsoon season in India

This investigation was performed to monitor hexachlorocyclohexane isomers (HCHs), dichlorodiphenyltrichloroethane (DDT, and its metabolites, refered to as DDTs), plasma levels of estradiol-17β (E2), and the gonadosomatic index (GSI) between sampling sites of unpolluted ponds of Gujartal, Jaunpur (control site) and the polluted rivers Gomti (Jaunpur) and Ganga (Varanasi), which affect the reproductive physiology of some edible catfish and carp during the pre-monsoon season. HCHs and DDTs were measured by gas liquid chromatography (GLC) and hormones by radioimmunoassay (RIA). The results indicated that the level of HCHs and DDTs was very high in both the catfish and the carp captured from the polluted rivers compared with the fish captured from the control site. The GSI and E2 values were lower in both groups of fish when compared to the fish from the control site. The results also indicate that catfish showed greater bioaccumulation of HCHs and DDTs than carp, above the permissible limit, as compared to the fish from the control site. In conclusion, fish from the Gomti and Ganga rivers were highly polluted when compared with fish from the control site, as was evident from high levels of tissue bioaccumulation of HCHs and DDTs and decreased levels of plasma E2, inhibiting the reproductive physiology of these species at the receptor level. The levels exceeded the maximum residue limits (MRL) as recommended by Codex, hence it is suggested that the fish should be avoided for food purposes.     

Influence of cortisol, growth hormone, insulin-like growth factor I and 3,3′,5-triiodo-l-thyronine on hypoosmoregulatory ability in the euryhaline teleost Fundulus heteroclitus

The capacity of cortisol, ovine growth hormone (oGH), recombinant bovine insulin-like growth factor I (rbIGF-I) and 3,3,5-triiodo-l-thyronine (T₃) to increase hypoosmoregulatory capacity in the euryhaline teleost Fundulus heteroclitus was examined. Fish acclimated to brackish water (BW, 10 ppt salinity) were injected with a single dose of hormone suspended in oil and transferred to seawater (SW, 35 ppt salinity) 10 days post-injection. Fish were sampled 24 h after transfer and plasma osmolality and gill Na⁺, K⁺-ATPase activity were examined. Transfer from BW to SW induced significantly increased plasma osmolality but not gill Na⁺, K⁺-ATPase activity. Cortisol (50 g g^–1 body weight) improved the ability to maintain plasma osmolality and to increase gill Na⁺, K⁺-ATPase activity. oGH (5 g g^–1 body weight) also increased hypoosmoregulatory ability and gill Na⁺, K⁺-ATPase activity. A cooperation between oGH and cortisol was observed in increasing hypoosmoregulatory ability but not in increasing gill Na⁺, K⁺-ATPase activity. rbIGF-I (0.5 g g^–1 body weight) alone was without effect in increasing salinity tolerance or gill Na⁺, K⁺-ATPase activity. rbIGF-I and oGH showed a positive interaction in increasing salinity tolerance, but not gill Na⁺, K⁺-ATPase activity. Treatment with T₃ (5 g g^–1 body weight) alone did not increase salinity tolerance or gill Na⁺, K⁺-ATPase activity, and there was no consistent significant interaction between cortisol and T₃ or between GH and T₃. The results confirm the classical role of cortisol as a seawater-adapting hormone and indicate an interaction between cortisol and the GH/IGF-I axis during seawater acclimation of Fundulus heteroclitus.     

The effects of hypoxia,in vivo,on red blood cell β-adrenoceptors in the rainbow trout,Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss, were exposed to 48h of environmental hypoxia (water partial pressure of oxygen = 8.0 kPa) at either 5 or 15°C. Blood was sampled during hypoxia via a dorsal aorta cannula to measure arterial blood partial pressure of oxygen and plasma catecholamine concentrations. After 48h, the number (B_max) and dissociation constant (K_d) of red blood cell surface -adrenoceptors were determined using a radioligand-displacement binding assay. At 5°C, plasma catecholamine levels were elevated at 24h whereas at 15°C, levels were elevated at 48h. At either temperature, following 48h of hypoxia, there was no change in B_max or K_d of red blood cell surface -adrenoceptors, compared to normoxic control fish. This study demonstrates that chronic exposure to moderate hypoxia does not affect the number or affinity of cell surface -adrenoceptors on trout red blood cells.     

Toxicity of wild juvenile “komonfugu” Takifugu flavipterus in the Bay of Sanriku Coast,Tohoku Area,Northern Japan

To clarify the mechanism of tetrodotoxin (TTX) accumulation in pufferfish, we compared the toxicity of two sets of wild juvenile “komonfugu” Takifugu flavipterus. The first set was sampled from Onisawa Fishing Port (FP) located in Okirai Bay, the Pacific Coast of Sanriku, Tohoku Area, Northern Japan. The second set was collected from the Onisawa FP and reared in an outdoor laboratory tank supplied with different seawater (Yoshihama Bay). The fish were sampled regularly and on the same days. The amount of TTX (mouse unit (MU)/fish) in the fish at Onisawa FP increased until 20 days and thereafter it did not change, while the amount of TTX in the fish in the laboratory tank remained low, and the TTX concentration (MU/g fish) decreased. Next, we compared the toxicity of wild juvenile T. flavipterus collected from Okirai Bay (Onisawa FP and Okirai FP) and Yoshihama Bay (Yoshihama FP). Large differences in TTX levels were observed among the fish from the three FPs. The amounts and concentrations of TTX in the fish at Onisawa FP were higher than those in the fish from the other two FPs. These results indicate that a large variation in toxic activity exists in the juvenile T. flavipterus in the bay of the Sanriku Coast.

     

The presence of 17α,20β-dihydroxy-4-pregnen-3-one receptor activity in the ovary of the brook trout,Salvelinus fontinalis,during terminal stages of oocyte maturation

The presence of 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) oocyte receptor activity has been demonstrated in brook troutSalvelinus fontinalis. Scatchard analyses of the cytosol fraction during various terminal stages of oocyte maturation gave a high equilibrium association constant (K_a) value of 1.394±0.669 10⁸M^–1 (n=7) and low maximum binding capacities (N_max). The association kinetics of the receptor was second order k₊₁=2.292×10⁶M^–1 sec^–1. The dissociation rate constant k_a was 1.502×10^–2 sec^–1 for the first order dissociation reaction. The K_a=1.526×10⁸M^–1, when it was determined from k₊₁/k_–1 a value close to that found from the Scatchard analysis. Competition studies showed the following binding affinities testosterone > 17-HP > 17,20-DHP > Promegestone > progesterone > estradiol > pregnenolone; cortisol showed no competitive inhibition. Cytosolic extracts when pre-equilibrated with various labelled steroids and eluted from a Sephacryl S-300 column gave multiple specific binding peaks. On sucrose density gradient centrifugation specific binding was observed at 3.05 S in cytosol containing 0.15M sodium chloride buffer. The receptor lost binding activity when incubated with various proteases, but DNase and RNase had no effect. Blood plasma without heparin at (110) dilution also bound [³H]17,20-DHP, Ka was 8.04×10⁷ M^–1.The nuclear pellet extract (750×g) gave very little specific binding activity even at high radiolabelled steroid concentrations and a linear Scatchard plot was not obtained. Nevertheless the nuclear extract, after dextran-charcoal treatment, pre-equilibrated with [³H]17,20-DHP, bound specifically to DNA cellulose, and cytosol from the same oocytes also bound to DNA cellulose under similar conditions. Although specific binding to DNA cellulose was obtained the salt concentrations at which the steroid-receptor complex elution took place was not reproducible in both nuclear extracts and cytosol samples. Also binding activity was extremely small compared to the total cytosolic binding. The nuclear extract when pre-equilibrated with high concentrations (20 nM) of the labelled steroid and then chromatographed on Sephacryl S-300 column gave a specific binding peak which was similar to that of the cytosolic preparation.The receptor levels in cytosol decreased progressively during final maturation (Stages 1–7). There is preliminary evidence for the presence of 17,20-DHP receptor activity in cytosol of landlocked Atlantic salmonSalmo salar ouananiche, and rainbow troutSalmo gairdneri. The zona radiata fraction from late stages oocyes 5, 6, and 7 in brook and rainbow trout oocytes were isolated by ultracentrifugation; from this fraction a protein was characterized which covalently bound [³H]R5020 after photoaffinity labelling. The same protein also bound [³H]17,20-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [³H]17,20-DHP, although the molecular weights were different. The blood sample [³H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17,20-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.A. Maneckjee is presently NSERC postgraduate scholar at MSRL and Ph.D. candidate at Department of Biochemistry, Memorial University of Newfoundland.     

Small rivers,great importance: Refuge and growth sites of juvenile migratory fishes in the upper São Francisco Basin,Brazil

Studies of migratory fish species in Neotropical Basins have generally focused on adult fishes, especially on their reproductive migration, whereas sites of growth and refuge of juveniles remain poorly investigated. We aimed to evaluate if smaller rivers play a role in the life cycle of these species. We used 13 rivers of different sizes in the upper São Francisco Basin, Brazil. We found that smaller rivers, especially when draining regions close to floodplains, were the first places where juveniles moved after leaving floodplain lakes. We also found that individuals moved downstream as they grew, and were found in increasingly larger rivers that were more distant from floodplains. Currently, the best-known strategy for conservation of neotropical migratory fishes is the maintenance of free-flowing stretches encompassing necessary habitats for life cycle completion. We conclude that small tributaries near floodplain regions are also needed in these protected free-flowing river systems.     

The “tsunami break:” impact of the Great East Japan Earthquake and the accompanying tsunamis on the shell growth of the invasive clam-eating snail <Emphasis Type="Italic">Laguncula pulchella</Emphasis>

The Great East Japan Earthquake and accompanying tsunamis occurred on 11 March 2011, causing huge damage to marine organisms. The invasive naticid gastropod Laguncula pulchella (Euspira fortunei), which was introduced with the imported clam Ruditapes philippinarum from China and Korea, survived the earthquake. The “growth break line” observed on the shell surface in over 90% of the individuals collected in Matsushima Bay and Matsukawa-ura Lagoon after the tsunamis was examined by scanning electron microscopy. Each shell presented three layers before and three after the growth break. However, the fracture surface consisted of five layers—two prismatic and three crossed lamellar—around the growth break line. This suggests that shell formation temporarily ceased following the tsunamis and that the five-layered shell may have developed in response to the stress caused by the tsunamis. The newly formed middle layer became thinner after the “tsunami break,” which may be the result of a rapid change in the mineralization process, including rapid shell growth and/or repair. These results suggest that the damage to and forcible removal of habitats by the tsunamis was stressful for L. pulchella. A decrease in or the cessation of shell formation after a tsunami may be a common phenomenon in mollusks that inhabit tidal flats.     

The effects of gonadal development and sex steroids on growth hormone secretion in the male tilapia hybrid (Oreochromis niloticus × O. aureus)

Profiles of plasma growth hormone (GH) in male tilapia hybrid (Oreochromis niloticus x O. aureus) were measured and compared at different times of the year. The profiles did not appear to be repetitive, however, differences in their nature were observed at the different seasons; the most erratic profiles were seen in the height of the reproductive season (July), while the peaks were more subdued in the spring and disappeared in the autumn. Peaks in male fish were more prominent than in the females when measured in July. Perifused pituitary fragments from fish with a high GSI responded to salmon gonadotropin-releasing hormone (sGnRH) analog (10 nM-1 M), while those from fish with a low GSI barely responded to even the highest dose. Exposure of perifused pituitary fragments from sexually-regressed fish to carp growth hormone-releasing hormone (cGHRH; 0.1 M) or sGnRH (I M) stimulated GH release only after injection of the fish with methyl testosterone (MT; 3 injections of 0.4 mg kg ¹). The same MT pretreatment did not alter the response to dopamine (DA; 1 or 10 M). GH pituitary content in MT-treated fish was lower than in control fish, which may be explained by the higher circulating GH levels in these fish, but does not account for the increased response to the releasing hormones. Castration abolished the response of cultured pituitary cells to sGnRH (I fM-100 nM) without altering either their basal rate of secretion or circulating GH levels. Addition of steroids to the culture medium (MT or estradiol at 10 nM for 2 days) enabled a GH response to sGnRH stimulation in cells from sexually regressed fish. Pituitary cells which had not been exposed to steroids failed to respond to sGnRH, although their response to forskolin or TPA was similar to that of steroid-exposed cells. It would appear, therefore, that at least one of the effects of the sex steroids on the response to GnRH is exerted proximally to the formation of cAMP, or PKC, presumably at the level of the receptor. An increase in the number of receptors to the GH-releasing hormones, following steroid exposure, would explain also the changing nature of the GH secretory profile in different stages of the reproductive season.     

Comparing the efficacy of dietary α-tocopherol with that of dl-α-tocopheryl acetate, both either alone or in combination with ascorbic acid, on growth and stress resistance of angelfish, Pterophylum scalare, juveniles

This study aimed to compare the efficacy of dietary α-tocopherol with that of dl-α-tocopheryl acetate, both either alone or in combination with vitamin C (ascorbic acid), on the growth performance, survival, and stress resistance of angelfish, Pterophylum scalare, juveniles. Juveniles were fed ad libitum for four weeks with Artemia enriched with no vitamins (control), vitamin C (T_c), α-tocopherol (T_α), dl-α-tocopheryl acetate (T _dl ), α-tocopherol and vitamin C (T_α+C), and dl-α-tocopheryl acetate and vitamin C (T _dl+C). After four weeks, an osmotic stress test was performed using seawater (25 g/L) to evaluate juvenile’s resistance to stress. Whole-body glucose and cortisol were used as stress indicators. At the end of the feeding trial, growth performance and survival of the juveniles fed vitamin-enriched Artemia were significantly (P < 0.05) higher than for the control fish. Best performance was recorded for the T_α+C group. Survival, however, was not significantly (P > 0.05) different between the vitamin-fed groups. Osmotic stress significantly elevated the stress indicators, whole-body cortisol and glucose levels (P < 0.05), highest and lowest values being observed in control and T_α+C groups, respectively. Survival after osmotic stress of juveniles fed the T_α+c diet was significantly higher (by 46.2%, P < 0.001) than for controls. Results suggested that α-tocopherol has greater efficacy than dl-α-tocopheryl acetate and enriching Artemia with α-tocopherol and vitamin C together improves growth performance, survival, and stress resistance of angelfish juveniles.     

Kinetic study of hepatic β-glucuronidase in the Indian major carp, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton)

Liver is the metabolic factory and contains several valuable enzymes that catalyze biochemical reactions. β-glucuronidase is one of the well-known lysozymes that participates in the carbohydrate metabolism in the tissues of various vertebrates. In the present study, an attempt was made to study the kinetic properties of hepatic β-glucuronidase of the Indian major carp (IMC), Labeo rohita. It was observed that the enzyme activity increased largely at pH 5 (0.1 M acetate buffer) when exposed to 38°C. However, the maximum activity was noticed at 52°C and later it started declined up to 70°C. It was also observed that with time the enzyme activity increased until substrate was completely used up. It has been concluded that it is a heat stable enzyme and cannot be destroyed at room temperature. Enzyme activity was observed to increase in response to increase in enzyme and substrate concentrations. The reaction velocity maxima (Vmax) and Michaelis constant (Km) were recorded using Lineweaver–Burk plot that was 18.182 μg/h and 2.907 mM, respectively.     

Inclusion of β-1,3/1,6-glucan in the ornamental fish,Jewel tetra (Hyphessobrycon eques), and its effects on growth,blood glucose,and intestinal histology

The effects of β-1,3/1,6-glucan on Hyphessobrycon eques were assessed after 42 days of feeding diets containing 0 (control group given commercial feed), 0.5, 1, or 2 g β-glucan/kg diet. In total, 180 fish, with an initial weight of 0.43?±?0.03 g, were used. There were 15 fish in each of twelve 42-L aquariums, and there were 3 aquariums of fish for each dietary treatment. The fish were fed until apparent satiety. Performance parameters (final weight, total length, standard length, feed intake, survival rate, weight gain, feed conversion, specific growth rate, and condition factor) and plasma glucose concentration were measured. Histological analysis of the proximal portion of the intestine (width and height of the villi, depth of the crypts, height of the enterocytes, thickness of the muscle layer, and number of goblet cells) was performed. Different levels of the additive did not influence fish performance (for example, final weight: control: 0.63 g, 0.5: 0.60, 1: 0.58, and 2: 0.61). Likewise, there was no influence on the plasma glucose concentration (control: 81.80 mg/dL, 0.5: 75.33, 1: 85.00, and 2: 81.00) and intestinal morphometry of the animals. However, the results showed that 2.0 g/kg of β-1,3/1,6-glucan provided a greater abundance of goblet cells secreting acidic and neutral mucus present in the epithelium (periodic acid-Schiff: 66.67 cells, Alcian blue pH 1.0: 72,67 cells, and Alcian blue pH 2.5: 95.00 cells), showing significant differences when compared to animals in the control group, which may represent better protection of the intestinal epithelium of H. eques.

     

Effects of salinity, dietary level of protein and 17α-methyltestosterone on growth hormone (GH) and prolactin (tPRL177 and tPRL188) levels in the tilapia, Oreochromis mossambicus

In the present study, we examined the long-term effects of environmental salinity, diet (35% and 25% crude protein) and 17-methyltestosterone (MT) on corresponding levels of pituitary and serum growth hormone (GH) and prolactins (tPRL₁₇₇ and tPRL₁₈₈) in the tilapia (Oreochromis mossambicus). We observed no discernible patterns in serum GH that would suggest an effect of salinity, diet or MT. However, serum GH levels in all treatments declined at 1 and 3h after first feeding. Serum tPRL₁₇₇ and tPRL₁₈₈ were significantly higher in freshwater (FW) than in seawater (SW) and levels were significantly affected by dietary protein. tPRL₁₇₇ levels were higher in all groups fed a 35% protein diet, but tPRL₁₈₈ levels were higher only in the groups fed the MT-treated 35% protein diet; only serum tPRL₁₈₈ levels were affected by MT. Moreover, serum tPRL₁₇₇ and tPRL₁₈₈ increased throughout the sampling time-course. Subsequent work using fasted tilapia suggests that first feeding is likely to initiate the post-prandial suppression of serum GH levels. In contrast with the picture observed in blood, pituitary glands of SW animals showed higher levels of GH than FW fish. Pituitary GH was elevated by MT in both FW and SW. We also observed that pituitary tPRL₁₇₇ and tPRL₁₈₈ levels were higher in FW fish than in SW fish; tPRL₁₇₇ and tPRL₁₈₈ levels were elevated by MT only in FW animals. To assess the somatomedin activity of plasma from FW- and SW-reared tilapia, we measured [35S]-sulfate incorporation into ceratobranchial cartilage explants in vitro. Plasma from SW-adapted tilapia showed greater activity in this assay than plasma from FW-reared tilapia, suggesting that the GH-dependent IGF bioactivity of plasma is higher in SW-reared tilapia. Collectively, these studies suggest that the growth-promoting actions of SW rearing and of MT administration in tilapia may be linked to elevations in GH and/or prolactin (tPRL₁₇₇ and tPRL₁₈₈)levels.     

Is the establishment of MPAs enough to preserve endangered intertidal species? The case of Patella ferruginea in Mal di Ventre Island (W Sardinia,Italy)

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Effect of diethylstilbestrol (DES) and 17 β-estradiol (E2) on growth, survival and histological structure of the internal organs in juvenile European catfish Silurus glanis (L.)

The effect of supplemented commercial diets with diethylstilbestrol (DES—15, 30 and 60 mg kg^?1) and 17 β-estradiol (E2—30 and 60 mg kg^?1), two chemicals commonly used in sex reversal procedure in fish, on survival and growth parameters of juvenile European catfish (Silurus glanis) was evaluated. During the two experiments, lasting 28 days each, fish were kept at temperature 25.2–26.5 °C, pH 7.4–9.3 and oxygen concentration 5.0–7.3 mg O₂ dm^?3. DES supplementation resulted in depressed growth rate of catfish. In experimental groups fed with E2, no negative effect on growth parameters was found. Both chemicals did not result in observed mortality. In all of the experimental DES groups, hepatosomatic index increased significantly, which suggests negative influence on physiological condition of catfish. DES supplementation significantly changed cytological factors of liver cells and caused hepatic alterations in parenchyma, such as vacuolization and blood congestion. Similarly, supplementation of E2 in food resulted in changes in cytological parameters of hepatocytes. However, E2 did not cause pathological changes within the liver tissue. Histological examination of the catfish gonads showed 19 and 38 % of sterile fish after treatment with 30 and 60 mg kg^?1 of DES, respectively. The results suggest that DES served in food could be ineffective in hormonal feminization process of European catfish. No disturbances of sex differentiation process after E2 treatment were observed. However, slight feminization effect in the highest level of E2 treatment group was recorded.     

The GABA a agonist muscimol enhances locomotor activity, but does not alter the behavioural effects of CRH in juvenile spring chinook salmon (Oncorhynchus tshawytscha)†

The present study investigated the effects of manipulating the GABAergic system on locomotor activity in juvenile spring chinook salmon, Oncorhynchus tshawytscha. In addition, we evaluated whether the GABAergic system is important for mediating the behavioural effects of corticotropin-releasing hormone (CRH). An intracerebroventricular (ICV) injection of the GABA_a agonist muscimol caused an acute and dose dependent increase in locomotor activity in juvenile spring chinook salmon. ICV injections of the GABA_a antagonist bicuculline prevented the increase in activity when administered concurrently with muscimol. The GABA_b agonist baclofen had no effect on locomotor activity in this study. Furthermore, we found no evidence that the locomotor response to exogenous CRH was altered by the concurrent administration of muscimol or bicuculline. These results provide evidence to support the hypothesis that endogenous GABA within the central nervous system is involved in the control of locomotor activity in fish. The data also suggest that there is no interaction between the GABAergic system and CRH with regards to the control of locomotor activity in this species.