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.     

Econometric model of viability/profitability of octopus (<Emphasis Type="Italic">Octopus vulgaris</Emphasis>) ongrowing in sea cages

The interest in diversifying aquacultural production with new species is evident, preferably with species with a high commercial value and whose consumption is not geographically limited. In this sense, octopus would be a good choice. The present work, therefore, presents an economic-financial analysis of the commercial viability of octopus ongrowing in the Mediterranean, paying special attention to the one- or two-cycle approach. Viability–profitability equations are developed for analysing economic parameters associated with production. To estimate the investment, an offshore ongrowing installation was designed comprising 150 cages containing 30,000 individuals. Growth was estimated for the two strategies: (A) Two consecutive cycles per year (2CY), each lasting 3.5 months from October to June. Initial weight was 0.7 kg, and the mean final weight was 2.7 kg. (B) One growth cycle per year (1CY) beginning in November or December and finishing in April or May, starting with the same weight individuals (0.7 kg) and giving individuals with a final weight of 3.65 kg. The highest costs, the most important from an economic point of view, are feed (38.57 and 40.03%, respectively), fixed assets (25.26 and 17.47%, respectively), juveniles (16.65 and 23.02%, respectively), and in fourth place salaries (14.34 and 15.60%, respectively). The equations obtained for the variables NPV (Net Present Value) and IRR (Internal Ratio of Return) are the following: 1 cycle per year (1CY), NPV = −489,088 − 1.45 K − 1,439,823 C _F − 1,477,890 C _J − 1,460,627 C _O + 1,432,386 SP, IRR = 0.1328 − 7.82 × 10⁻⁸ K − 0.0416 C _F − 0.0437 C _J − 0.0427 C _O + 0.0412 SP. 2 cycles per year (2CY), NPV = −404,431 − 1.46 K − 2,118,410 C _F − 2,121,221 C _J − 2,144,755 C _O + 2,129,223 SP, and IRR = 0.0952 − 6.95 × 10⁻⁸ K − 0.0586 C _F − 0.0588 C _J − 0.0588 C _O + 0.0613 SP. The NPV and IRR values estimated with the econometric equations for each option using the initial variables confirm that 1CY has a higher NPV (3,013,569 €) and IRR (12.27%) than 2CY, with an NPV of 2,396,708 € and IRR of 10.39%. In both cases, octopus ongrowing is economically viable, although 1CY is the most favourable system.     

Bioaugmentation in the growth and water quality of livebearing ornamental fishes

Bacillus subtilis isolated from the intestine of Cirrhinus mrigala (Hamilton) was incorporated into the rearing water of Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) at four different concentrations (5 × 10⁸ cells ml⁻¹, 5 × 10⁷ cells ml⁻¹, 5 × 10⁶ cells ml⁻¹ and 5 × 10⁵ cells ml⁻¹) and its effect on fish growth performance and survival, water quality parameters and bacterial population of water were assessed. The results showed that the addition of bacterial cells in the rearing water resulted in greater survival and a faster growth rate and, hence, greater length and weight increments of the livebearers. The use of a bioaugmentor in the rearing water of the livebearing fishes resulted in significantly lower (P < 0.05) concentrations of dissolved organic matter and total ammonium nitrogen. The counts of motile aeromonads and total coliforms recorded in the water of bioaugmented tanks were also lower than that in the control tank. Bioaugmentation between 10⁶ and 10⁸ cells ml⁻¹ in the rearing water is sufficient in establishing a bioaugmentor and the use of a higher concentration of bacterial cells did not always lead to significantly better results.     

Trypsin from the viscera of Bogue (<Emphasis Type="Italic">Boops boops</Emphasis>): isolation and characterisation

Trypsin from the viscera of Bogue (Boops boops) was purified to homogeneity by precipitation with ammonium sulphate, Sephadex G-100 gel filtration and Mono Q-Sepharose anion exchange chromatography, with an 8.5-fold increase in specific activity and 36% recovery. The molecular weight of the purified enzyme was estimated to be 23 kDa by SDS–PAGE and size exclusion chromatography. The purified trypsin appeared as a single band on native-PAGE and zymography staining. The purified enzyme showed esterase-specific activity on N-α-benzoyl-l-arginine ethyl ester (BAEE) and amidase activity on N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity, after 10 min incubation, were pH 9.0 and 55°C, respectively, using BAPNA as a substrate. The trypsin kinetic constants K _m and k _cat on BAPNA were 0.13 mM and 1.56 s⁻¹, respectively, while the catalytic efficiency k _cat /K _m was 12 s⁻¹ mM⁻¹. Biochemical characterisation of B. boops trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish processing and food industries.     

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.     

Effect of light intensity on self-feeding of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> reared individually

The self-feeding abilities of individually reared rainbow trout Oncorhynchus mykiss were investigated under four different light intensities (7×10⁻², 1×10°, 5×10¹, and 7×10² lx). There was no significant effect of light intensity on self-feeder learning ability and food wastage (which did not exceed 0.5%). However, there was high individual variability in the ability irrespective of light intensity. The total number of trigger actuations was significantly higher at 1×10° lx than at 7×10⁻² and 5×10¹ lx. Feeding patterns during the light phase of a day were classified as early or late in relation to the timing of significant peaks in fish feeding, or non-steady. The last pattern was defined when significant peaks were not observed. Under 7×10⁻², 1×10°, or 7×10² lx, a non-steady feeding pattern was the most common. Early feeding appeared only under the 1×10° lx regime with the random pattern, while the late pattern was observed under 7×10⁻², 5×10¹, and 7×10² lx. Under 5×10¹ lx regime half of the fish fed in the afternoon. These results suggest that light intensity can affect the timing of feeding judged from the frequency of pulling bite tags, but does not affect waste or learning period.     

Effect of temperature on α-tocopherol, fatty acid profile, and pigments of Diacronema vlkianum (Haptophyceae)

Diacronema vlkianum was grown in polyethylene bags at two different temperatures (18 and 26°C) in the laboratory. The biochemical composition level decreased when the temperature increased from 18 to 26°C. The maximum cell number at 18°C was 11.9 × 10⁶ cells ml⁻¹, while maximum cell number at 26°C was 1.6 × 10⁶ cells ml⁻¹. The maximum level of α-tocopherol was 257.7 ± 21.6 μg g⁻¹ dry weight (DW) at 18°C. The highest total carotenoids and chlorophylls were 6.5 mg g⁻¹ DW and 4.3 mg g⁻¹ DW, respectively, and the main pigments were determined as astaxanthin and lutein. Polyunsaturated fatty acids were found to be the predominant group, reaching 39.5% of the total fatty acids at 18°C. This comprised 20:5(n − 3) as the main polyunsaturated fatty acids (20.4%, at 18°C) followed by 22:6(n − 3) (4.8%, at 18°C). The results suggest that D. vlkianum can be successfully used as feed in shellfish hatcheries or aquaculture hatcheries, either as a substitute or in association with other microalgae, when this algae is cultured at 18°C.     

Migration history of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis> captured in the Sea of Okhotsk,northern Japan,using otolith Sr:Ca ratios

Sakhalin taimen Hucho perryi populations have decreased drastically to near extinction. It is urgent to establish an effective conservation strategy based on an understanding of the characteristics of migration and habitat use of this species. We examined the migration history of anadromous Sakhalin taimen captured off the Sarufutsu coast, northern Hokkaido, Japan, using otolith Sr:Ca ratios and also examined the relationship between their otolith Sr:Ca ratios during freshwater and seawater residence in a rearing experiment. Otolith Sr:Ca ratios of some fish from the Sarufutsu coast showed freshwater levels (0.5–4.0 × 10⁻³) near the core, which thereafter increased to brackish water levels (4.0–6.0 × 10⁻³), and then to seawater levels (6.0–10.0 × 10⁻³) in the outermost regions. Those findings indicate that specimens from the Sarufutsu coast migrated to the brackish water region or the sea and spent most of their lives there. The anadromous migration pattern including the timing of downstream migration seems to be flexible among individuals in the species. They migrate between freshwater and seawater or brackish water several times during their lives, showing extensive habitat use. It is essential to secure the continuity among the freshwater, brackish water, and seawater areas for their effective conservation.     

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.     

Immunolocalization of chloride transporters to gill epithelia of euryhaline teleosts with opposite salinity-induced Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase responses

Opposite patterns of branchial Na⁺/K⁺-ATPase (NKA) responses were found in euryhaline milkfish (Chanos chanos) and pufferfish (Tetraodon nigroviridis) upon salinity challenge. Because the electrochemical gradient established by NKA is thought to be the driving force for transcellular Cl⁻ transport in fish gills, the aim of this study was to explore whether the differential patterns of NKA responses found in milkfish and pufferfish would lead to distinct distribution of Cl⁻ transporters in their gill epithelial cells indicating different Cl⁻ transport mechanisms. In this study, immunolocalization of various Cl⁻ transport proteins, including Na⁺/K⁺/2Cl⁻ cotransporter (NKCC), cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 1 (AE1), and chloride channel 3 (ClC-3), were double stained with NKA, the basolateral marker of branchial mitochondrion-rich cells (MRCs), to reveal the localization of these transporter proteins in gill MRC of FW- or SW-acclimated milkfish and pufferfish. Confocal microscopic observations showed that the localization of these transport proteins in the gill MRCs of the two studied species were similar. However, the number of gill NKA-immunoreactive (IR) cells in milkfish and pufferfish exhibited to vary with environmental salinities. An increase in the number of NKA-IR cells should lead to the elevation of NKA activity in FW milkfish and SW pufferfish. Taken together, the opposite branchial NKA responses observed in milkfish and pufferfish upon salinity challenge could be attributed to alterations in the number of NKA-IR cells. Furthermore, the localization of these Cl⁻ transporters in gill MRCs of the two studied species was identical. It depicted the two studied euryhaline species possess the similar Cl⁻ transport mechanisms in gills.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

Shrimp shell as a potential sorbent for removal of arsenic from aqueous solution

This study examined the ability of shrimp shell to remove arsenic (As) from aqueous solutions. The shells of two species of shrimp, black tiger shrimp Penaeus monodon and white shrimp Litopenaeus vannamei, were chosen to be the sorbents. Laboratory exposure experiments estimated uptake and depuration rate constants (i.e., k ₁ and k ₂) as well as the bioconcentration factors (BCF) of the shells of the two shrimps. A first-order one-compartment model was presented to describe the uptake kinetics of As in shrimp shell. The resulting k ₁, k ₂, and BCF values of black tiger shrimp were 0.034–1.722 ml/g/day, 0.007–0.345 g/g/day, and 5.08 ± 1.56 ml/g, while those for white shrimp were 0.053–0.523 ml/g/day, 0.011–0.237 g/g/day, and 3.95 ± 1.88 ml/g, respectively. The sorption capacities of black tiger shrimp shell and white shrimp shell were 1.08 × 10⁻⁴–6.66 × 10⁻³ and 1.04 × 10⁻⁴–3.26 × 10⁻³ mmol/g, respectively. The sorption capacity of shrimp shell increased with the initial As concentration in water. Shrimp shell, as a waste material, could be potentially used for the removal of As from an aqueous medium. Although the As-removal capacity of shrimp shell was lower than those of natural and chemical sorbents, using shrimp shells as sorbents is less expensive and could increase the additional value of shrimp products.     

Occurrence of estuarine and sea eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and a migrating silver eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> in the Tokyo Bay area,Japan

Otolith microchemical analyses of the strontium (Sr) and calcium (Ca) concentrations in the eels Anguilla japonica and A. anguilla caught in Tokyo Bay were undertaken to reconstruct the eels’ migratory histories. A. japonica in the yellow stage (immature stage) were caught in a bay without any adjacent rivers or streams. A. anguilla was in the silver stage (early maturing stage), and the eel was confirmed to have just begun spawning migration to the open ocean from Tokyo Bay based on the otolith Sr:Ca ratios, which showed a typical catadromous life history with low Sr:Ca ratio values throughout the eel’s life after recruitment. The mean Sr:Ca ratios in A. japonica from the elver mark to the otolith edge indicated the eels belonged to several general categories of migratory histories, including sea eels (average Sr:Ca ratio ≥6.0 × 10⁻³) and estuarine eels (average Sr:Ca ratio 2.5 to 6.0 × 10⁻³) based on the criteria reported previously in A. japonica. All eels had a certain freshwater life period, although the period was highly variable among fish. These results indicate that A. japonica has a flexible pattern of migration, with the ability to adapt to various habitats and salinities.     

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.     

Characterization of Pseudomonas aeruginosa associated with diseased postlarval abalone in Shenzhen, China

Outbreaks of mass mortality of postlarval abalone, Haliotis diversicolor supertexta, have occurred in south China since 2002 and have forced many abalone farms to close. About 30 representative bacterial strains were isolated from a sample of five diseased postlarval abalone, taken 25 days post-fertilization during an outbreak of postlarval disease in Shenzhen, China, in October 2006. Bacterial challenge tests showed that the predominant strain, designated as strain 22, was highly virulent to postlarvae with an LD₅₀ value of 7.8 × 10⁴ colony forming units (CFU) ml⁻¹, while four of the other isolates were weakly virulent with LD₅₀ values ranging from 1 × 10⁶ to 1 × 10⁷ CFU ml⁻¹, and the remaining 25 isolates were classified as avirulent with LD₅₀ values greater than 1 × 10⁸ CFU ml⁻¹. By means of API 20NE and 16S rDNA and ITS sequencing analyses, strain 22 was identified as Pseudomonas aeruginosa. Antibiotic susceptibility tests showed that strain 22 exhibited around 75% of susceptibility to 16 various antibiotics tested. The results of this study show P. aeruginosa as one of the bacteria involved in the mortality of abalone postlarvae in Shenzhen, China.     

Growth,diet, and reproduction of Eurasian perch <Emphasis Type="Italic">Perca fluviatilis</Emphasis> L. in Lake Varese,northwestern Italy

No data have previously been reported on Eurasian perch Perca fluviatilis L. in Lake Varese. In this study, the growth, diet, and reproductive biology of the Eurasian perch population were investigated with the aim of providing information that may serve as a basis for efficient resource management. A total of 240 specimens were caught during the monthly sampling campaign from November 2006 through October 2008. The length-to-weight relationships were W _t = 8.4 × 10⁻³ L _t^3.10 (males) and W _t = 4.1 × 10⁻³ L _t^3.36 (females). The parameters for the von Bertalanffy growth function for pooled sexes were L _∞  = 33.17 cm, k = 0.20 year⁻¹, and t ₀ = −1.34 year. Perch in Lake Varese spawn from April through May. Sexual maturity is reached when males are 2 years old, in females mostly when they are 3 years old. Relative fecundity (F _rel) and absolute fecundity (F _abs) were assessed for females. Fecundity values were similar to data reported for other European populations: females of age 2+ F _rel = 102,457 ± 12,275, age 3+ F _rel = 131,767 ± 5,891, and age 4+ F _rel = 131,252 ± 15,555. Perch diet spectrum was wide and somewhat characterized by season. Perch in Lake Varese feed on macroinvertebrates, mainly Chironomidae and Chaoborus, zooplankton, and juvenile rudd Scardinius erythrophthalmus.     

Comparative growth of Pacific oyster (Crassostrea gigas) postlarvae with microfeed and microalgal diets

Two trials were carried out in the laboratory in order to assess the effect of microparticulated feed (F) and live (Thalassiosira pseudonana, M) diets on the growth of recently set (396 ± 13 μm shell height) and 2 mm Crassostrea gigas postlarvae. Different proportions of M and F (100:0, 75:25, 50:50; 25:75, 0:100) were delivered in a single dose of 3 h d⁻¹ in trial 1. Dietary M:F proportions of 100:0, 50:50, and 0:100 were delivered as a single pulse of 8 h d⁻¹ (P1) or two pulses of 4 h⁻¹ (P2) in trial 2. Maximal daily M ration was 296 cells μl⁻¹ d⁻¹ (trial 1), 150 M cells μl⁻¹ d⁻¹ (trial 2), or their equivalent F dry weight. Shell height (SH), dry (DW), and organic weight (AFDW) were evaluated weekly. Oysters from trial 1 significantly increased their size after 28 days, and exhibited no significant dietary differences in terms of DW (1.21 ± 0.15 to 2.01 ± 0.28 mg) or AFDW (0.091 ± 0.022 to 0.166 ± 0.029 mg). Newly set postlarvae (trial 2) also exhibited significant growth after 25 days. No dietary differences were observed in trial 2, yet P2 oysters attained significantly higher shell heights (825–912 μm) than P1 oysters (730–766 μm) after 25 d. Pulse effects were marginally not significant in terms of AFDW and growth rate. Together, these findings showed that balanced microfeeds have a practical potential for the culture of early C. gigas postlarvae, when they are delivered in pulse-feeding schemes     

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.     

Effect of prebiotic xylooligosaccharides on growth performances and digestive enzyme activities of allogynogenetic crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>)

The effect of prebiotic xylooligosaccharides (XOS) on the growth performance and digestive enzyme activities of the allogynogenetic crucian carp, Carassius auratus gibelio, was investigated. XOS was added to fish basal semi-purified diets at three concentrations by dry feed weight: diet 1, 50 mg kg⁻¹; diet 2, 100 mg kg⁻¹; diet 3, 200 mg kg⁻¹, respectively. Twelve aquaria (n = 20) with three replicates for each treatment group (diets 1–3) and control treated without XOS were used. Weights of all collected carp from each aquarium were determined at the initial phase and at the end of the experiment, and the carp survival was also determined by counting the individuals in each aquarium. After 45 days, there were significant differences (P < 0.05) in the relative gain rate (RGR), and daily weight gain (DWG) of diets 1–3 were compared with the control. However, the survival rate was not affected (P > 0.05) by the dietary treatments. For enzymatic analysis, dissection produced a crude mixture of intestine and hepatopancreas of each segment to measure. The protease activity in the intestine and hepatopancreas content of fish in diet 2 (487.37 ± 20.58 U g⁻¹ and 20.52 ± 1.93 U g⁻¹) were significantly different (P < 0.05) from that in the control (428.13 ± 23.26 U g⁻¹ and 12.81 ± 1.52 U g⁻¹) and diet 3 (428.00 ± 23.78 U g⁻¹ and 14.04 ± 1.59 U g⁻¹). Amylase activity in the intestine was significantly higher for diet 2 compared to diet 1 and the control. As for amylase in the hepatopancreas, assays showed higher activity in diet 2 (P < 0.05) compared to the rest.     

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm⁻²) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H _O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.