Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.     

Prevention of thaw-rigor during frozen storage of bigeye tuna Thunnus obesus and meat quality evaluation

Thaw-rigor is often found in frozen meat of bigeye tuna Thunnus obesus. Excessive amounts of drip loss and stiffness greatly lower the commercial value of tuna meat. In order to prevent thaw-rigor in meat stored at −60°C post-capture, we adapted a temperature shift technique that stores the meat at −7°C for 1 day or −10°C for 7 days before thawing. Biochemical changes in muscle of bigeye tuna before and after the temperature shift to −7 or −10°C were characterized. Contents of ATP, NAD⁺, glycogen, and creatine phosphate decreased after the temperature shift. NAD⁺ levels decreased faster than ATP levels and were highly correlated with the rigor index. Thaw-rigor occurred in muscle containing NAD⁺ at 1 μmol/g and ATP at 7 μmol/g. On the other hand, the meat color of tuna during frozen storage changed to brown depending on the storage temperature and reflected the rate of metmyoglobin (met-Mb) formation. Met-Mb formation increase was dependent on the decrease in NADH levels during the frozen storage. A temperature shift technique with storage at −7°C for 1 day or −10°C for 7 days before thawing prevented thaw-rigor and met-Mb formation.     

拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10⁻⁷ and 1.05 × 10⁻⁷ M, respectively, by a quartz crystal microbalance technique.     

Study on the distribution and expression of a DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

The distribution and expression of lymphocystis disease virus (LCDV) vaccine, on the basis of DNA vaccine (pEGFP-N2-LCDV0.6 kb) construction, were analyzed in tissues of the Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR studies indicated that the vaccine-containing plasmids were distributed in injected muscle, muscle located opposite the injection site, hind intestine, gill, spleen, head kidney, liver and gonad 7 days after vaccination. However, these vaccine-containing plasmids disappeared by 90 days following vaccination. Fluorescent microscopy observations revealed that green fluorescence appeared in muscle, muscle located at the opposite side of the injection site, hind intestine, gill, spleen, head kidney and liver of fish 36 h after vaccination, and that green fluorescence did not appear in control tissue. The green fluorescence became weaker at 60 days post-vaccination, however, it remained detectable in the spleen 90 days post-vaccination. Results from RT-PCR studies indicated that the Mcp gene is expressed in all tissues of vaccinated fish 7-20 days after vaccination. These results demonstrate that the DNA vaccine is distributed and expressed in different tissues of vaccinated fish, and therefore, may have provided an antigen producing specific immune response.     

The effect of energy reserves and cryoprotectants on overwintering mortality in Mercenaria mercenaria notata (Say 1822) at two tidal levels

The objective of this study is to identify possible causes of the high winter mortality noted in juvenile Mercenaria mercenaria notata in eastern Canada. The percentage mortality, shell growth, concentration of energy reserves, and production of cryoprotectant molecules were compared between notata and native quahogs kept at intertidal and subtidal levels. Overwintering mortality of the notata strain reached 47.2% and was 3–9 times higher than that of the native strain. Shell increment was higher in the native than in the notata strain and also at the intertidal than at the subtidal level. The quahogs from the subtidal zone had a higher concentration of protein than those from the intertidal zone in August and April but a lower concentration in February. The notata strain had a lower concentration of lipids and glucose (34.9 and 0.21 mg g⁻¹ dry weight) than the native strain (42.2 and 0.28 mg g⁻¹ dry weight). Thermal hysteresis was detected in none of the quahog groups. High winter mortality in the notata strain seems to be caused, in part, by a lower capacity to stock lipid compared with the native strain. The higher concentration of glucose in the native strain may favour survival in cold water by helping to reduce the freezing point of the animals’ fluids.     

Swimming physiology of European silver eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.): energetic costs and effects on sexual maturation and reproduction

The European eel migrates 5,000–6,000 km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10–12 mg fat/km which is 4–6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61–0.67 m s⁻¹, which is ~60% higher than the generally assumed cruise speed of 0.4 m s⁻¹ and implies that female eels may reach the Sargasso Sea within 3.5 months instead of the assumed 6 months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.     

Dietary protein requirement for fingerling Channa punctatus (Bloch), based on growth, feed conversion, protein retention and biochemical composition

An 8-week feeding trial was conducted in a flow-through system (1–1.5 L min⁻¹) at 27°C to determine dietary protein requirement for Channa punctatus fingerlings (4.58 ± 0.29 g) by feeding six isocaloric diets (18.39 kJ g⁻¹, gross energy). Diets containing graded levels of protein (300, 350, 400, 450, 500 and 550 g kg⁻¹) were fed to triplicate groups of fish to apparent satiation at 09:00 and 16:00 h. Maximum absolute weight gain (AWG; 8.11 g fish⁻¹), specific growth rate (SGR; 1.82%) and best feed conversion ratio (FCR; 1.48) were recorded in fish fed diet containing 450 g kg⁻¹ protein, whereas protein efficiency ratio (PER; 1.52), protein retention efficiency (PRE; 25%), energy retention efficiency (ERE; 78%) and RNA/DNA ratio (3.01) were maximum for the group fed dietary protein at 400 g kg⁻¹. Second-degree polynomial regression analysis of AWG, SGR and FCR data against varying levels of dietary protein yielded optimum dietary protein requirement of fingerling between 462.24 and 476.72 g kg⁻¹, whereas the regression analysis of PER, PRE, ERE and RNA/DNA ratio data showed a lower protein requirement of 438.28–444.43 g kg⁻¹ of the diet. Considering the PER, PRE, ERE and RNA/DNA ratio as more reliable indicators, this protein requirement is recommended for developing quality protein commercial feeds for C. punctatus fingerlings.     

Optimum ration level for better growth,conversion efficiencies and body composition of fingerling <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis> (Bloch)

The effects of ration levels on growth, conversion efficiencies and body composition of fingerling Heteropneustes fossilis (6.8 ± 0.04 cm, 5.0 ± 0.02 g) were studied by feeding isonitrogenous (40% crude protein) and isocaloric (19.06 MJ kg⁻¹ gross energy) diets representing 1, 3, 5, 7 and 9% of the body weight (BW) day⁻¹ to triplicate groups of fish . Growth performance of the fish fed at the various ration levels was evaluated on the basis of live weight gain percentage (LWG%), feed conversion ratio (FCR), specific growth rate percentage (SGR%), protein retention efficiency (PRE%) and energy retention efficiency (ERE%) data. Maximum LWG% and SGR were obtained at a feeding rate of 7% BW day⁻¹, whereas best FCR (1.6), PRE% and ERE% were recorded at a feeding rate of 5% BW day⁻¹. Maximum body protein was also obtained for the group receiving the diet representing 5% of their body weight. However, a linear increase in fat content was noted with the increase in ration levels up to 7% BW day⁻¹. The SGR, FCR, PRE and ERE data were also analyzed using second-degree polynomial regression analysis to obtain more precise information on ration level, with the results showing that the optimal ration for these parameters was 6.8, 6.1, 5.9 and 6.2% BW day⁻¹, respectively. Based on the above second-degree polynomial regression analysis, the optimum ration level for better growth, conversion efficiencies and body composition of fingerling H. fossilis was found to be in the range of 5.9–6.8% of the BW day⁻¹, corresponding to 2.36–2.72 g protein and 88.20–101.66 MJ digestible energy kg⁻¹ diet day⁻¹.     

Transgenic medaka with brilliant fluorescence in skeletal muscle under normal light

ABSTRACT:   Green fluorescent protein ( GFP ) and red fluorescent protein ( RFP ) genes regulated by the medaka skeletal muscle actin promoter were microinjected into fertilized d-rR medaka eggs to establish transgenic medaka lines. Intense fluorescence was detected in skeletal muscle. During development, GFP and RFP became detectable in anterior somites at the 12- and 30-somete stages, respectively. After hatching, intense fluorescence in skeletal muscle enabled individual fish to be identified under normal lighting without fluorescent microscopy. Fluorescence was also observed in the gills and esophagus of the adult fish. These data indicated that medaka lines are convenient not only for the study of skeletal muscle but also for the identification of cells or individuals in various studies.     

Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (<Emphasis Type="Italic">Monopterus albus</Emphasis> Zuiew)

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish rice field eel (Monopterus albus Zuiew) by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephacryl S-200 HR. The molecular masses of the three purified PGs were all estimated as 36 kDa using SDS–PAGE. Two-dimensional gel electrophoresis (2D-PAGE) showed that pI values of the three PGs were 5.1, 4.8, and 4.6, respectively. All the PGs converted into corresponding pepsins quickly at pH 2.0, and their activities could be specifically inhibited by aspartic proteinase inhibitor pepstatin A. Optimum pH and temperature of the enzymes for hydrolyzing hemoglobin were 3.0–3.5 and 40–45°C. The K _m values of them were 1.2 × 10⁻⁴ M, 8.7 × 10⁻⁵ M, and 6.9 × 10⁻⁵ M, respectively. The turnover numbers (k _cat) of them were 23.2, 24.0, and 42.6 s⁻¹. Purified pepsins were effective in the degradation of fish muscular proteins, suggesting their digestive functions physiologically.     

Protein-sparing effect of dietary lipid in practical diets for blunt snout bream (Megalobrama amblycephala) fingerlings: effects on digestive and metabolic responses

This study aimed to evaluate the protein-sparing effect of dietary lipid on digestive and metabolic responses of fingerling Megalobrama amblycephala. Fish were fed nine practical diets with three protein levels (270, 310 and 350 g kg⁻¹) and three lipid levels (40, 70 and 100 g kg⁻¹) for 8 weeks. Weight gain was significantly affected only by dietary lipid levels with the highest found in fish fed 70 g kg⁻¹ lipid. Relative feed intake and whole-body protein content showed little difference among all the treatments. Activities of intestine lipase and amylase increased significantly as dietary lipid levels increased, whereas little difference was observed in protease activities. Liver lipid content was significantly affected only by protein levels with the lowest found in fish fed 310 g kg⁻¹ protein. Liver aspartate aminotransferase (GOT) activities increased significantly with decreasing lipid levels, whereas the highest GOT activity was obtained in fish fed 310 g kg⁻¹ protein in terms of dietary protein levels. Activities of liver lipoprotein lipase, total lipase and plasma cholesterol concentration of fish fed 350 g kg⁻¹ protein were significantly lower than that of the other groups, whereas the same was true for plasma 3, 5, 3′-triiodothyronine level of fish fed 270 g kg⁻¹ protein. The results indicated that an increase of dietary lipid content from 40 to 70 g kg⁻¹ can enhance the growth and digestive enzyme activities of this species and reduce the proportion of dietary protein catabolized for energy without inducing hepatic steatosis; meanwhile, decreasing protein level from 350 to 310 g kg⁻¹ leads to the increase of lipase activities both in intestine and liver coupled with the reduced liver lipid content.     

The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes

The effects of porcine, scombroid, and salmon insulins, and bovine and anglerfish glucagons on glycogen depletion and glycogen phosphorylase (GPase) activities were examined in freshly isolated American eel (Anguilla rostrata) hepatocytes. Eel liver GPase in crude homogenates was activated (increase in % GPase a) by phosphorylating conditions and was rapidly inactivated (less than 1 h) when a phosphatase inhibitor (fluoride) was absent. Caffeine inhibits, and AMP activates, the b form of GPase consistent with their effects on rat liver GPase. Both mammalian and fish glucagons increased glucose production in eel hepatocytes, but had more ambiguous effects on glycogen levels and GPase activities. The magnitude of bovine glucagon effects were dependent on the initial glycogen content of the cells; only at glycogen concentrations less than approximately 70 μmoles.g⁻¹ did glucagon significantly increase % GPase a. Anglerfish glucagon significantly increased cyclic AMP (cAMP) concentrations by 90% at 10⁻⁷ M, but had no effects at 10⁻⁹ M and 10⁻⁸ M. Scombroid and salmon insulins maintained hepatocyte glycogen concentrations and decreased glucose production, with these effects more pronounced at low (10⁻⁹ to 10⁻⁸ M) rather than high (10⁻⁷ M) hormone concentrations. Porcine and salmon insulins decreased total GPase and % GPase a activities, and salmon insulin decreased CAMP levels, but only at 10⁻⁸ M (by 44%). Glycogen is, therefore, depleted by glucagon and maintained by insulin in freshly isolated American eel hepatocytes, and these changes are accomplished, at least in part, by changes in the activities of GPase. Changes in cAMP do not explain all of the observed hormone effects.     

Effect of long-term administration of dietary β-1,3-glucan on growth, physiological, and immune responses in Litopenaeus vannamei (Boone, 1931)

β-1,3-Glucan at different dietary doses was administered to enhance the growth, immunity, and survival against nitrite stress in Pacific white shrimp, Litopenaeus vannamei. Four different diets supplemented with 0, 250, 500, or 1,000 mg of β-1,3-glucan kg⁻¹ diets were fed to L. vannamei. Growth performance (weight gain and survival rate), physiological conditions (blood total protein, glucose, lactate, triacylglycerols, cholesterol levels) and immunological responses (superoxide dismutase, catalase, lysozyme, acid phosphatase, and alkaline phosphatase activities) of shrimp were recorded after 84-day feeding and 120 h after exposed to nitrite-N. After 84-day feeding, 250 mg kg⁻¹ β-1,3-glucan diet resulted in better weight gain (P < 0.05). Before the nitrite stress, blood lactate, triacylglycerols, and cholesterol level in shrimp fed with 250 mg kg⁻¹ β-1,3-glucan diet were significantly higher than those observed in shrimp fed with other diets (P < 0.05). Higher activities of catalase, lysozyme, and alkaline phosphatase were observed in shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan diet as compared to those obtained in shrimp fed with other diets (P < 0.05). After 120-h nitrite stress, blood protein, lactate, superoxide dismutase, catalase, and alkaline phosphatase activities in shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan were significantly higher than those observed in shrimp fed with other diets (P < 0.05). Glucose and triacylglycerol levels of shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan were significantly lower than those observed in other diets (P < 0.05). In shrimp fed with 500 and 1,000 mg kg⁻¹ β-1,3-glucan and 120-h after nitrite stress, the mortality was significantly lower than that observed in shrimp of control. Together, in this 84-day feeding trial, 250 mg kg⁻¹ β-1,3-glucan improved growth, whereas 500 mg kg⁻¹ β-1,3-glucan preferentially improved nitrite resistance, probably through accelerating energy metabolism and activating immune system.     

Low fishmeal diets for Atlantic salmon, <Emphasis Type="Italic">Salmo salar</Emphasis> L., using soy protein concentrate treated with graded levels of phytase

The experiment aimed at determining the efficient use of phytase (Phy) in Atlantic salmon diets that had low (4.5%) fishmeal and contained 60% soy protein concentrate (SPC). Phytase was either included at 250, 500, 1,000 or 4,000 U Phy kg⁻¹ diet or the SPC was pre-treated prior to making diets using 250, 500 or 1,000 U Phy kg⁻¹ SPC. Fish were fed the experimental diets for 12 weeks, and there were no differences in survival among treatments nor were there differences in growth performance between the phytase-pre-treated SPC diets. Feed intake and weight gain were significantly lower for diets supplemented below 1,000 U Phy kg⁻¹ compared to all other diets. Apparent digestibility (AD) of phosphorus was significantly lower without the use of phytase (45.43 ± 2.06%) than for all other treatments. AD phosphorus increased from 55.70 ± 1.81% at the lowest phytase supplementation (250 U Phy kg⁻¹) to 80.87 ± 2.12% at the highest (4,000 U Phy kg⁻¹). There was no difference in AD phosphorus between the diet with the highest supplementation (4,000 U Phy kg⁻¹) and the pre-treated diets. There were no differences in whole-body dry material, crude protein or total lipid, whereas bone ash was significantly lower for diets supplemented below 1,000 U Phy kg⁻¹. Ash and phosphorus in the whole body and bone increased with increasing added phytase. At and above an inclusion of 1,000 U Phy kg⁻¹, bone ash (51.26 ± 0.12% bone weight) and bone phosphorus (11.21 ± 0.04% bone weight) reached concentrations that were no different to the pre-treated diets. In conclusion, phytase improved Atlantic salmon’s growth performance fed low fishmeal diets containing SPC, and at least 1,000 U Phy kg⁻¹ diet was required to have the same effect as pre-treatment of SPC with 250 U Phy kg⁻¹ SPC.     

Cod CGRP and tachykinins in coeliac artery innervation of the Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>: presence and vasoactivity

The presence and vasoactive effects of native calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) were studied on isolated small branches of the coeliac artery from Atlantic cod, Gadus morhua, using immunohistochemistry and myograph recordings, respectively. Immunohistochemistry revealed nerve fibers containing CGRP- and SP/NKA-like material running along the wall of the arteries. CGRP induced vasorelaxation of precontracted arteries with a pD₂ value of 8.54 ± 0.17. Relaxation to CGRP (10⁻⁸ M) was unaffected by l-NAME (3 × 10⁻⁴ M) and indomethacin (10⁻⁶ M) suggesting no involvement of nitric oxide or prostaglandins in the CGRP-induced relaxation. SP and NKA (from 10⁻¹⁰ to 3 × 10⁻⁷ M) contracted the unstimulated arteries at concentrations from 10⁻⁸ M and above in 42% and 33%, respectively, of the vessels. It is concluded that the innervation of the cod celiac artery includes nerves expressing CGRP-like and tachykinin-like material, and that a vasodilatory response to CGRP is highly conserved amongst vertebrates while the response to tachykinins is more variable.     

Benthic nutrient fluxes at longline sea squirt and oyster aquaculture farms and their role in coastal ecosystems

To assess the impact of aquaculture activities, we measured the primary production, total sediment oxygen consumption, and benthic nutrient flux at two aquaculture farms (sea squirt and oyster) and one reference site. The estimated primary production in the water column ranged from 19–169 mmol C m⁻² d⁻¹. Total sediment oxygen consumption rates ranged from 58 to 328 mmol m⁻² d⁻¹, 1.1 to 40 mmol m⁻² d⁻¹ for nitrogen, 0.17 to 3.0 mmol m⁻² d⁻¹ for phosphate, and 7.3 to 74 mmol m⁻² d⁻¹ for silicate. The average total sediment oxygen consumption at the longline farms was >2.5 times higher than the reference site. Nitrate was significantly removed by denitrification at the longline farms in July and September and ranged from −5.4 to −0.09 mmol m⁻² d⁻¹, which is higher than for other coastal sediments. The benthic fluxes of nitrogen and phosphate at longline farms were up to 16 and six times higher than at the reference site, respectively. The average nitrogen requirements of the primary producers were 9.3 mmol m⁻² d⁻¹ at the sea squirt farm, 7.0 mmol m⁻² d⁻¹ at the oyster farm, and 13.5 mmol m⁻² d⁻¹ at the reference site, corresponding to 88, 316, and 27.2% of the nitrogen supplied by benthic fluxes, respectively. Our results suggest that benthic nutrient fluxes at longline farms are a major nutrient source for primary production in coastal waters.     

Metabolic adaptations of oxidative muscle during spawning migration in the Atlantic salmon <Emphasis Type="Italic">Salmo salar</Emphasis> L.

The adaptability/plasticity of the highly oxidative red muscle in Atlantic salmon was demonstrated during spawning migration. Substrate concentrations and the enzymatic pathways of ATP production were examined in red muscle obtained from Atlantic salmon at different sites along their migratory route in the Exploits River, Newfoundland, Canada. Individuals were chronologically sampled from a seawater site, two sites upstream, and at spawning. The 20% decrease in salmon body weight during the later stages of migration was accompanied by large decreases (mg dry weight⁻¹) in both glycogen (P < 0.01) and total muscle lipid (P < 0.01). In contrast, water content and protein concentration (mg dry weight⁻¹) of the red muscle increased by 25 and 34%, respectively, at spawning. Enzymes of the glycolytic pathways demonstrated a significant (P < 0.001) decrease in maximal activity as migration proceeded whereas enzymes of the oxidative phosphorylation pathways, specifically the citric acid cycle enzymes, exhibited an increase (P < 0.001) in maximal activity at spawning. The antioxidant enzyme superoxide dismutase also demonstrated an increase (P < 0.001) in maximal activity during the latter stages of migration. These adaptations imply that the red epaxial muscle of Atlantic salmon has a more efficient means of oxidizing lipids, while minimizing free radical damage, during the later stages of migration and spawning, thereby potentially increasing post spawning survival.     

Biochemical and pharmacological characterization of adenosine A1 receptors in eel (Anguilla anguilla) brain

N⁶-cyclohexyl[³H]adenosine ([³H]CHA) was used to label adenosine A1 receptors in membranes prepared from male and female eel whole brain. The A1 receptor agonist [³H]CHA bound saturably, reversibly and with high affinity (K_d = 0.91 ± 0.12 nM; B_max = 120.36 ± 5.2 fmol mg⁻¹ protein). In equilibrium competition experiments, the adenosine agonists and antagonists all displaced [³H]CHA from high-affinity binding sites with the rank order of potency in displacing, characteristics of an A1 adenosine receptor. Mg²⁺ dramatically increased the affinity of [³H]CHA without modifying the maximal binding capacity. The specific binding was inhibited by guanosine 5′-triphosphate (K_i = 2.54 ± 0.98 μM). The [³H]CHA binding sites are ubiquitously distributed with a maximum in cerebellum and a minimum in olfactory bulb. No difference was observed between male and female brain. In eel brain, synaptosomes (P2), stimulation of adenosine 3′,5′-monophosphate (cyclic AMP) accumulation with 10⁻⁵ M forskolin was markedly reduced (45.5%) by treatment with the adenosine A1 receptor agonist CHA (10⁻⁴ M), and the reduction was reversed in presence of the selective A1 receptor antagonist 8-cyclopentyltheophylline (10⁻⁵ M). In superfused eel cerebellar synaptosomes, K⁺ stimulated the release of adenosine in a partially Ca²⁺-dependent manner. The findings, taken together, suggest the hypothesis that adenosine A1 receptors present in eel brain could modulate synaptic transmission, as A1 receptors do in other vertebrates.     

Difference Between Tetrodotoxin and Saxitoxins in Accumulation in Puffer Fish Takifugu rubripes Liver Tissue Slices

In vitro accumulation of tetrodotoxin (TTX) and paralytic shellfish toxin (PST) in tiger puffer fish Takifugu rubripes was investigated using liver tissue slices. When T. rubripes liver slices were incubated with Leibovitz’s L-15 medium containing 0.13 mM TTX at 20 °C in air with saturated humidity, they accumulated 21.5 ± 7.3 μg TTX g⁻¹ liver after the incubation for 12 h and increased to 55.3 ± 8.2 μg TTX g⁻¹ liver at 48 h. In the incubation of T. rubripes liver slices with 0.13 mM PST-containing medium, PST was detected 6.3 ± 0.9 μg g⁻¹ liver at 12 h and reached a plateau thereafter. These results reveal the difference between TTX and PST in accumulation in T. rubripes liver tissue slices. To examine the variation in PST accumulation among fish species, the liver tissue slices from tiger puffer fish T. rubripes, parrot-bass Oplegnathus fasciatus and green ling Hexagrammos otakii were incubated at a concentration of 0.027 mM PST. The toxin contents of 3.0 μg g⁻¹ liver were observed at 8 h regardless of fish species but were not increased subsequently, showing no variety among these three species as to accumulation patterns of PST. It is noted that the tiger puffer fish T. rubripes liver specifically accumulate TTX in preference to PST.