miR-462-731对草鱼线粒体功能的影响

硬骨鱼特有的miR-462与miR-731位于同一基因簇(简写为miR-462-731),在低氧环境中表达量会显著上调。为了进一步研究miR-462-731簇的作用,本研究以草鱼(Ctenopharyngodon idella)肝脏细胞为对象,分析miR-462与miR-731过表达对细胞线粒体生物学功能的影响。结果显示miR-462与miR-731过表达后三羧酸循环中关键基因mdh、ogdh、cs的mRNA表达水平以及ATP含量均显著降低,线粒体膜电位下降;细胞中活性氧(reactiveoxygen species,ROS)的含量显著增加,同时总抗氧化能力(totalantioxidantcapacity,T-AOC)及超氧化物歧化酶(superoxide dismutase,SOD)活性显著下降,而丙二醛(malondialdehyde,MDA)含量升高;电子显微镜观察发现miR-462与miR-731过表达后细胞线粒体结构损伤。由此推测,miR-462-731簇可通过影响细胞线粒体膜电位、代谢及氧化应激等参与线粒体活动,研究结果可为草鱼及其他硬骨鱼类低氧调节机制提供基础资料。     

Physiological stress responses in spotted wolffish (Anarhichas minor) subjected to acute disturbance and progressive hypoxia

The spotted wolffish (Anarhichas minor) is considered a promising species for coldwater aquaculture. It is a sedentary, bottom-dwelling fish which exhibits a calm and “non-stressed” behaviour in captivity. There are, however, no reports on the physiological responses to stressors in this species. In the present study we investigated primary (cortisol secretion) and secondary (glucose mobilization) responses to common aquaculture stressors like disturbance and hypoxia. Pre-stress plasma cortisol levels were within those generally considered representative for unstressed fish (~ 10 ng ml^− 1), whereas basal glucose levels were unusually low (0.3–0.4 mM). After exposure to a short, but severe disturbance challenge (emptying the tank of water for 10 min), the increase in plasma cortisol level was slow and relatively weak, reaching a peak level of 25 ng ml^− 1 4 to 8 h after disturbance. When the fish were exposed to a gradually decreasing oxygen level in the tank, a significantly elevated plasma cortisol level (35 ng ml^− 1) was seen in the fish that remained in the tank until oxygen saturation had decreased to 20% oxygen saturation after 2.5 h. However, a two-fold higher plasma cortisol level (~ 70 ng ml^− 1) was seen in all fish exposed to reduced oxygen levels (60, 40 and 20% oxygen saturation) after 3.5 h recovery in normoxic water. Plasma glucose levels showed only moderate increases (~ 70%) following disturbance and hypoxia challenges. An in vivo injection of ACTH caused a strong elevation of plasma cortisol (peak level ~ 170 ng ml^− 1), demonstrating a high capacity for interrenal steroidogenesis in the spotted wolffish. The slow and relatively weak cortisol response to stressors, and low plasma glucose levels, may relate to the sedentary lifestyle of the spotted wolffish. The stress-response is characterised by a passive (reactive) coping style, which is considered adaptive for farming of this species.     

Assessment of tissue-specific effect of cadmium on antioxidant defense system and lipid peroxidation in freshwater murrel, Channa punctatus

The effects of different concentrations of cadmium chloride on the extent of lipid peroxidation (LPO) and alterations in the antioxidant enzyme activities were studied in liver, kidney and gill tissues of freshwater murrel, Channa punctatus. The fish specimens were exposed to 6.7, 13.4 and 20.1 mg l⁻¹ sublethal concentrations of cadmium chloride and the oxidative stress was assessed after 24, 48, 72 and 96 h post-exposure. The biomarkers selected for the study were thiobarbituric acid reactive substances for assessing the extent of lipid peroxidation and antioxidant defense system such as reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GP_X), glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) activities. In general, the cadmium exposure elevated the LPO in subject tissues of treated group and modulated the activities of GPx, GST, SOD, CAT, GR and level of GSH after given exposure as compared to the control. All enzymes activities, except CAT (in kidney and gills), and amount of LPO elevated significantly (P < 0.05) in treated group with respect to control in all tissues, while significant difference was not observed between the exposed concentrations and within exposure duration. The results indicated that increase in LPO level and the fluctuation in antioxidant defense system in fish could be due to cadmium-induced increase in the production of reactive oxygen species (ROS). The potential role of these parameters as biomarkers of heavy metal pollution in aquatic system is discussed.     

The capacity of oysters to regulate energy metabolism‐related processes may be key to their resilience against ocean acidification

Bivalve molluscs, such as oysters, are threatened by shifts in seawater chemistry resulting from climate change. However, a few species and populations within a species stand out for their capacity to cope with the impacts of climate change‐associated stressors. Understanding the intracellular basis of such differential responses can contribute to the development of strategies to minimise the pervasive effects of a changing ocean on marine organisms. In this study, we explored the intracellular responses to ocean acidification in two genetically distinct populations of Sydney rock oysters (Saccostrea glomerata). Selectively bred and wild type oysters exhibited markedly different mitochondrial integrities (mitochondrial membrane potential) and levels of reactive oxygen species (ROS) in their hemocytes under CO₂ stress. Analysis of these cellular parameters after 4 and 15 days of exposure to elevated CO₂ indicated that the onset of intracellular responses occurred earlier in the selectively bred oysters when compared to the wild type population. This may be due to an inherent capacity for increased intracellular energy production or adaptive energy reallocation in the selectively bred population. The differences observed in mitochondrial integrity and in ROS formation between oyster breeding lines reveal candidate biological processes that may underlie resilience or susceptibility to ocean acidification. Such processes can be targeted in breeding programs aiming to mitigate the impacts of climate change on threatened species.     

Spectrofluorimetric study of the interaction of methyl-parathion with fish serum albumin

The interaction of methyl-parathion with the albumin of Piaractus mesopotamicus (Holmberg 1887) (= pacu), a fish species typical of Brazilian rivers, was studied and the results compared with known values for human and bovine albumin obtained in an earlier investigation. Methyl-parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) is an organophosphorous pesticide still used in agriculture and fish farming in many countries. The fluorescence quenching technique with tryptophan as a natural probe was used to detect for the presence of methyl-parathion. Fluorescence can be mathematically expressed by the Stern–Volmer equation to calculate quenching constants, and changes in the behavior of Stern–Volmer curves at different temperatures indicate the nature of the mechanism causing the quenching. Our results indicate that methyl-parathion forms a complex with fish albumin. The estimated association constant is 9.73 × 103 (± 4.9 × 102) M⁻¹ at 25°C.     

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.     

Effects of temperature and salinity on oxygen consumption and ammonia excretion of juvenile miiuy croaker, <Emphasis Type="Italic">Miichthys miiuy</Emphasis> (Basilewsky)

The metabolic responses of the juvenile Miichthys miiuy in terms of oxygen consumption and ammonia excretion to changes in temperature (6–25°C) and salinity (16–31 ppt) were investigated. At a constant salinity of 26 ppt, the oxygen consumption rate (OCR) of the fish increased with an increase in temperature and ranged between 133.38 and 594.96 μg O₂ h⁻¹ g⁻¹ DW. The effect of temperature on OCR was significant (P < 0.01). Q₁₀ coefficients were 6.80, 1.41, 1.29 and 2.36 at temperatures of 6–10, 10–15, 15–20 and 20–25°C, respectively, suggesting that the juveniles of M. miiuy will be well adapted to the field temperature in the summer, but not in the winter. The ammonium excretion rates (AER) of the fish were also affected significantly by temperature (P < 0.01). The O:N ratio at temperatures of 6, 10, 15 and 20°C ranged from 13.12 to 20.91, which was indicative of a protein-dominated metabolism, whereas the O:N at a temperature of 25°C was 51.37, suggesting that protein-lipids were used as an energy substrate. At a constant temperature of 15°C, the OCRs of the fish ranged between 334.14 (at 31 ppt) and 409.68 (at 16 ppt) μg O₂ h⁻¹ g⁻¹ DW. No significant differences were observed in the OCR and AER of the juveniles between salinities of 26 and 31 ppt (P > 0.05). The OCR and AER at 16 ppt were, however, significantly higher than those at 26 and 31 ppt (P < 0.05), indicating salinity lower than 16 ppt is presumably stressful to M. miiuy juveniles.     

Toxic effects of mercury chloride on silver catfish (Rhamdia quelen) spermatozoa

Heavy metals are highly toxic elements that are present in the environment, especially in water. Mercury chloride (HgCl₂) stands out among these compounds because of its strong ability to induce damage to any tissue with which it comes into contact. The gametes of spawning aquatic animals, such as fish, are susceptible to such damage. Thus, our objective was to evaluate the toxic potential of HgCl₂ in the capacitation and activation of Rhamdia quelen sperm. Semen was collected from seven males and activated in 58 mM sodium chloride (NaCl) containing 0 (control), 4^?10, 7^?10, 7^?9, and 7^?8 M HgCl₂. The evaluated variables included motility, vigor, motility time, morphology, membrane integrity, membrane fluidity, mitochondrial functionality, production of reactive oxygen species (ROS), and DNA fragmentation. All evaluated HgCl₂ concentrations increased primary pathologies and reduced motility, vigor and motility time. Damage to membrane integrity and fluidity began occurring at a concentration of 7^?10 M HgCl₂. These results indicate that HgCl₂ has a toxic effect on different sites of fish spermatozoa and that sperm motility decreases after exposure to HgCl₂, impairing sperm capacitation and activation.     

Effects of different natural or prepared diets on growth and survival of juvenile spider crabs, <Emphasis Type="Italic">Maja brachydactyla</Emphasis> (Balss, 1922)

The effects of different diets (natural or pellets) on growth, survival, and moulting interval of juvenile spider crabs, weighing between 0.011–1.56 g and up to 17.6 mm in carapace length, were tested over a period of 90 days. During experiment I, five diets were tested: (1) frozen shrimp—Paleomonetes sp., (2) fresh mussels—Mytilus sp., (3) white fish fillets—Merlucius merlucius, (4) blue fish fillets—Sardina pilchardus, and (5) commercial crustacean pellets. Spider crabs fed fresh mussels grew larger (0.98 ± 0.69 g) and had higher growth rates (4.0 ± 0.7 %BWd⁻¹) compared to the other four diets. The crabs fed shrimp pellet and frozen shrimp grew to intermediate sizes and were smaller than the ones fed fresh mussels, but they were larger than spider crabs fed either blue or white fish fillets (0.46 ± 0.63 and 0.26 ± 0.13 g, respectively) compared to the ones fed white fish fillets (0.12 ± 0.04) and blue fish fillets (0.04 ± 0.02 g). The spider crabs fed blue fish fillets only lasted until day 60 of the experiment, after this day none of the 20 fed this diet were left. During experiment II, two diets were tested: (1) white and blue fish fillets and (2) commercial fish pellet. There were no differences in growth both in weight or carapace length (2.9 ± 1.8 and 2.1 ± 1.5 g in weight, and 18.9 ± 5.0 and 17.7 ± 3.3 mm, respectively) at the end of the experiment. Similarly, there were no differences in growth rates in weight between the two diets (1.2 ± 0.4 and 0.9 ± 0.3 %BWd⁻¹, respectively) or in carapace length (0.4 ± 0.1 and 0.4 ± 0.2 %BWd⁻¹, respectively). Fresh mussel appears to be a very good diet to culture the early stages of this species, while shrimp pellets also deliver acceptable results. On the contrary, frozen shrimp, fish fillets either from blue or white species (much higher lipid content in the blue species), and fish pellets were found to be bad diets for the culture of the early stages of M. brachydactyla.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Acclimation of <Emphasis Type="Italic">Anabas testudineus</Emphasis> (Bloch) to three test temperatures influences thermal tolerance and oxygen consumption

Teleost fish have developed their own specific adaptive mechanism, both behavioral and physiological, to maintain homeostasis in response to unfavorable temperatures. Therefore, this study was aimed at assessing the critical thermal maxima (CT_Max), critical thermal minima (CT_Min), and oxygen consumption rate of Anabas testudineus (17.03 ± 1.2 g) after acclimating to three preset temperatures (25, 30, and 35°C) for 30 days. The CT_Max and CT_Min were 40.15, 41.40, 41.88°C and 12.43, 13.06, 13.94°C, respectively, and were significantly different (P < 0.05). The thermal tolerance polygon for the specified temperatures was 278.30°C². The oxygen consumption rate (117.03, 125.70, 198.48 mg O₂ kg⁻¹ h⁻¹, respectively) increased significantly (P < 0.05) with increasing acclimation temperatures. The overall results indicate that the thermal tolerance and oxygen consumption of A. testudineus are dependent on acclimation.     

Physiological studies on the effect of copper nicotinate (Cu–N complex) on the fish, <Emphasis Type="Italic">Clarias gariepinus</Emphasis>, exposed to mercuric chloride

Female catfish, Clarias gariepinus, were collected from the Nile River at Assiut region, were divided into 7 groups. The first group was left as control, and the second was treated with mercuric chloride (MC) for 3 weeks following by normal water for 1 week. The third, fourth and fifth groups were provided by MC (150 μg/ l of water). This treatment was continued for 3 weeks. Then, the fish were received CN instead of MC, for 1 week, with 15 and 25 mg CN/100 g wet food. The fifth fish group received diet supplemented with vit E (α-tocopherol) (100 mg/kg wet diet), for 1 week, instead of MC treatment. Vitamin E was used as standard antioxidant drug. Following 3 weeks of normal ambient water, the sixth and seventh aquaria received only CN for 1 week, with 15 and 25 mg CN respectively/100 g wet food, respectively. At the end of the experiment, Samples of liver, kidneys (posterior part), gills (right gills) and ovary were excised. The measurement included the oxidative stress parameters: carbonyl protein and total peroxide and the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in all selected organs. MC treatment induced harmful effect in fish, probably due to its enhancing effect on reactive oxygen species (ROS) production in fish organs especially the respiratory and osmoregulatory organs namely gills. The result suggests that this gill damage may exert hypoxic case, anoxia for different organs and some Cu excretion resulting in a magnification of ROS overproduction. Also, the observed oxidative stress in ovary tissue of MC-treated fish may affect fish fertility. The addition of CN in fish diets could protect the fish C. gariepinus against MC-induced oxidative damage showing recovery of fish organs. It could suggest that the detoxifying mechanism of action of CN is mainly due to its scavenging activity of free radicals rather than tissue healing.     

Purification and characterization of α<Subscript>1</Subscript>-proteinase inhibitor and antithrombin III: major serpins of rainbow trout (<Emphasis Type="Italic">Oncorhynchuss mykiss</Emphasis>) and carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) blood plasma

The main serine proteinase inhibitors of rainbow trout (Oncorhynchuss mykiss) and common carp (Cyprinus carpio) blood plasma were isolated and purified. The investigated inhibitors, α₁-proteinase inhibitor (α₁-PI) and antithrombin III (AT III), act by forming stable complexes with target proteinases. The association rate constants k _on for the interaction of fish plasma inhibitors with several serine proteinases have been determined: k _on for both carp and rainbow trout α₁-PI were >10⁷ M⁻¹ s⁻¹ for human neutrophil elastase, and in the case of bovine trypsin and chymotrypsin k _on values were 2.0–5.2 × 10⁶ M⁻¹ s⁻¹. Association rate constants k _on for the interaction of carp and rainbow trout AT III with bovine trypsin and thrombin were about 1.3 × 10⁴–7.9 × 10⁵ M⁻¹ s⁻¹ without and >10⁷ M⁻¹ s⁻¹ in presence of heparin; so antithrombins require the presence of heparin to become effective proteinase inhibitors. The high degree of homology of the estimated amino acid sequences of fish inhibitors reactive site loops confirms their similarity with other proteinase inhibitors from the serpin family.     

Effect of dietary chromium picolinate on growth performance and blood parameters in grass carp fingerling, <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>

An experiment was conducted to investigate the effect of dietary chromium picolinate supplement on growth and haematology parameters of grass carp, Ctenopharyngodon idellus. Six diets with increasing dietary chromium picolinate levels 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mg kg⁻¹ were fed to triplicate groups of 20 fish (initial weight of 12.78 ± 1.16 g, mean ± SD) in a flow water system for 10 weeks. Fish fed the diet supplemented with 0.8 mg Cr kg⁻¹ had significantly improved weight gain (WG), feed efficiency ratio (FER), protein efficiency ratio (PER) and protein retention (PR). Fish fed high-chromium diets exhibited lower whole-body crude lipid contents than fish fed low-chromium diets. Liver glycogen concentrations for fish fed the diet with 0.2 mg Cr kg⁻¹ was the highest (77.67 mg g⁻¹). Fish fed the diet supplemented with 1.6 and 3.2 mg Cr kg⁻¹ had significantly lower liver glycogen concentrations than other groups (P < 0.05). The highest serum insulin concentrations were observed in fish fed the diet supplemented with 0.8 mg Cr kg⁻¹, but serum insulin concentrations decreased (P < 0.05) when dietary supplementation of chromium was higher than 0.8 mg Cr kg⁻¹. Cholesterol concentrations decreased in direct proportion to dietary chromium level and achieved the lowest level when the fish were fed the 0.8 mg Cr kg⁻¹ diet, but increased when the fish were fed the diet with more than 0.8 mg Cr kg⁻¹ (P < 0.05). Fish fed the diet supplemented with 0.8 mg Cr kg⁻¹ had higher triglyceride and high-density lipoprotein cholesterol (HDL-C) concentrations compared to other treatments. The results of the present study suggested that chromium picolinate could modify serum carbohydrate and lipid metabolism profile, and that the optimal dietary chromium level was 0.8 mg kg⁻¹ for grass carp according to growth.     

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii

Vibrio ordalii is an extracellular, Gram‐negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo‐LM‐18 and ATCC 33509^T in the fish‐cell lines SHK‐1 and CHSE‐214. Confocal microscopy revealed Vo‐LM‐18 and ATCC 33509^T inside cytoplasm in both fish‐cell lines at 4 hr post‐inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo‐LM‐18 and ATCC 33509^T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.     

Reproductive dynamics and nursery habitat preferences of two commercially important Indo-Pacific red snappers <Emphasis Type="Italic">Lutjanus erythropterus</Emphasis> and <Emphasis Type="Italic">L. malabaricus</Emphasis>

Red snappers were examined for reproductive biology and age-0 habitat preferences. Spawning in red snappers occurred throughout the year in northern Australia and eastern Indonesia; at least 10–30% of females and 40–80% of males were in ripe or spawning condition in most months. Northern Australian populations showed a spawning peak from July to December (L. erythropterus) and September to March (L. malabaricus). Eastern Indonesian L. malabaricus had a less defined pattern with two peaks: January–March and October. Size at first maturity was 240 mm for males and 250–300 mm for females. L ₅₀ estimates were similar between species in northern Australia: 270–280 mm (males) and 350–370 mm (females). Maximum batch fecundity was 676,100 oocytes for L. erythropterus and 997,000 oocytes for L. malabaricus. Higher mean abundances of age-0 L. erythropterus were found in silty and coarse sand/rubble estuarine habitats of northern Australia (456 ± 119 fish/km²) compared with sandy coastal habitats (5 ± 3 fish/km²). Most age-0 snapper caught at Sape (eastern Indonesia) were L. malabaricus (91%) with mean abundances of 312 ± 14 fish/km². The similarities in the reproductive characteristics of red snappers suggest that successful management approaches adopted in northern Australia should be considered in eastern Indonesia.     

Quantification of relative flying fish paste content in the processed seafood ago-noyaki using real-time PCR

Real-time polymerase chain reaction (PCR) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence was used to determine flying fish paste in ago-noyaki. We quantified the amount of flying fish paste in ago-noyaki samples using flying fish-specific primers (Tobi16SF3/Tobi16SR) and universal primers (Univ16SF2/Univ16SR2). Using real-time PCR of standard ago-noyaki, a standard equation was obtained (y = 1.08x − 3.20; R ² = 0.977). This equation was then used to estimate the relative flying fish paste contents of eight commercially available ago-noyaki and two similar products. These results verified that the ago-noyaki products that had already been labeled with the E-mark deserved this status.     

Species identification of Alaska pollock,Gadus spp., and Micromesistius spp. in cod roe products using a PCR-based method

Fish species identification techniques for authentic food labeling were developed using species-specific PCR primers for cod roe products. A salted, seasoned fish roe product, karashimentaiko (chilli cod roe), is produced from the eggs of Alaska pollock, Theragra chalcogramma, according to the fair trade competition agreement authorized by the Fair Trade Commission of the Japanese government. To examine whether Alaska pollock ovaries or those of other fish species are being used as raw materials for the fish roe products, we developed species identification techniques using PCR amplification of a 255-bp fragment encoding the mitochondrial ATP synthase Fo subunit 6 (ATP6) gene with a species-specific primer set for Alaska pollock mitochondrial DNA. We also designed two species-specific primer sets corresponding to the mitochondrial ATP6 and cytochrome b (cytb) for Gadus spp. and Micromesistius spp. by PCR amplification of 332- and 223-bp fragments, respectively. We examined the species specificity of these PCR-based methods among nine commercially important Gadidae species.     

Oxygen consumption and ammonia excretion of black carp (Mylopharyngdon piceus Richardson) and allogynogenetic crucian carp (Carassius auratus gibelio ♀?×?Cyprinus carpio ♂) fed different carbohydrate diets

Oxygen consumption and ammonia excretion of black carp (Mylopharyngdon piceus Richardson) (4.6 ± 0.3 g) and allogynogenetic crucian carp (Carassius auratus gibelio ♀ × Cyprinus carpio ♂) (5.7 ± 0.5 g) were examined when fish fed two types of carbohydrate (dextrin and glucose) at two levels (20 and 40%) each. The diets were isonitrogenous (40% dry matter) and isocaloric at 18.5 kJ g⁻¹ (dry matter) by adjusting the oil content to 10.1 and 1.5%, respectively. In black carp, the interactions between the carbohydrate type and level were found in oxygen consumption at 3 and 6 h and in ammonia excretion at 6 h after feeding. At 20% carbohydrate, no significant difference was observed between dextrin and glucose in oxygen consumption. However, at 40% carbohydrate, oxygen consumption in fish fed glucose was significantly higher than that in fish fed dextrin at 3 and 6 h after feeding. Within the dextrin diets, no significant differences in both oxygen consumption and ammonia excretion were detected between the two carbohydrate levels. Within the glucose diets, however, fish fed 40% glucose showed significantly higher oxygen consumption than those fed 20% glucose at 3 and 6 h after feeding. Ammonia excretion in black carp fed 40% glucose was higher than that in black carp fed 40% dextrin at 6 h and also found higher than those in the other three treatments at 24 h after feeding. The postprandial oxygen consumption and the ammonia excretion in crucian carp fed 40% glucose were the highest, but no significant differences were observed. Our data indicate that the escalation of glucose to 40% in a fish diet results in high oxygen consumption and ammonia excretion in black carp, suggesting that the efficiency of glucose as an energy source for this fish is compromised by the high metabolic expenditure after feeding. Crucian carp, on the other hand, have a better ability to cope with dietary carbohydrates.