Development of methods for detection of SNPs in <Emphasis Type="Italic">Activin type IIB receptor</Emphasis> and a preliminary study on association between its polymorphism and growth parameters of the Asian seabass <Emphasis Type="Italic">Lates calcarifer</Emphasis>

Molecular markers that allow selection of juveniles and broodstock with a high growth performance are useful for the aquaculture industry. In this study, association between single nucleotide polymorphisms (SNPs) of the Activin type IIB receptor (LcActRIIB) and growth-related parameters of Asian seabass Lates calcarifer juveniles were examined by PCR-direct sequencing. Three SNPs (G>A_c.965+g.7, C>A>T _c.965+g.33 and G>A _c.965+g.189) generating seven composite SNP genotypes (diplotypes) were found in the LcActRIIB gene segment (398 bp). Among three identified SNPs, only a G>A_c.965+g.7 SNP showed significant association with growth traits (average body weight, total length, hepatic weight and hepatosomatic index) of examined fish (P < 0.01). Significant association between diplotypes and growth-related parameters were found. Juveniles exhibiting diplotypes I–V showed greater growth-related parameters than those exhibiting diplotypes VI (P < 0.001; N = 57). Simple methods for rapid genotyping of a G>A_c.965+g.7 SNP were successfully developed based on gel-based bi-allelic PCR amplification of specific alleles (Bi-PASA) and real-time PCR-based PASA analyses (N = 99). Identical genotypes were consistently obtained from different detection methods.     

Oocyte maturation and active motility of spermatozoa are triggered by retinoic acid in pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis>

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.     

Feeding,somatic condition and survival of sand lance <Emphasis Type="Italic">Ammodytes</Emphasis> sp. larvae in Mutsu Bay,Japan

To clarify the recruitment process of sand lance Ammodytes sp., we investigated larval condition factor, relative gut fullness (%GF), prey abundance and oceanographic structure in Mutsu Bay, Japan, during 1999–2001. Ammodytes sp. larvae, which were collected by horizontal hauls of Motoda nets and a ring net at depths of 1, 10, 20, 30 and 40 m, were mainly distributed at 10–30 m. Larvae at the first feeding time until 12 mm in body length (BL) fed predominantly on copepod nauplii, whereas large larvae with BL of 12.1–14.0 mm fed on a mixture of copepod nauplii, copepodites and appendicularians from late February to April. A path analysis showed that difference in water density between 35- and 5-m depths negatively affected naupliar abundance at 10–30-m depth (standardised path coefficient β = ?0.71, p = 0.005 for 3.3–8.0-mm BL larvae and β = ?0.78, p < 0.001 for 8.1–12.0-mm BL larvae). Naupliar abundance positively affected the %GF of Ammodytes sp. larvae (β = 0.75, p < 0.001 for 3.3–8.0-mm BL larvae and β = 0.66, p < 0.001 for 8.1–12.0-mm BL larvae), whereas it was negatively affected by water temperature (β = ?0.45, p = 0.008 for 3.3–8.0-mm BL larvae and β = ?0.56, p = 0.002 for 8.1–12.0-mm BL larvae), and the temperature effect was weak compared with that of naupliar abundance. In turn, %GF positively affected larval somatic weight (β = 0.91, p < 0.001 for 6.0-mm BL larvae and β = 0.70, p = 0.005 for 10.0-mm BL larvae). The recruitment failure in 1999 was likely caused by a reduced condition factor, which resulted from low naupliar abundance. In contrast, the abundance of nauplii and Oithona similis copepodites was high in 2000 and 2001. It is possible that the higher recruitment success in 2001 was because of the higher water temperatures in Mutsu Bay, sustaining faster growth of the larvae than in 2000 under the high-prey abundance conditions.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Growth performance and biochemical composition of juvenile pearl oyster <Emphasis Type="Italic">Pinctada martensii</Emphasis> fed on artificial diets

The effects of three artificial diets (S1, S2 and S3) on survival, growth and biochemical composition of one-year-old pearl oyster Pinctada martensii were investigated. Six experimental groups (EG1, EG2, EG3, EG4, EG5 and EG6) and one control group (CG) were set up. EG1, EG2 and EG3 were solely fed on S1, S2 and S3, respectively. EG4, EG5 and EG6 were fed on mixed diets, as follows: S1 and Platymonas subcordiformis; S2 and P. subcordiformis; and S3 and P. subcordiformis, respectively. CG was fed on only P. subcordiformis. All groups were continuously fed for 60 days. Survival, growth and biochemical composition of soft tissues were compared across the groups. Results showed that survival rate, the absolute growth rate (AGR) and relative growth rate (RGR) of shell length did not differ significantly across the groups (p > 0.05). The AGR and RGR of total weight differed significantly among the groups (p < 0.05). AGR and RGR of shell length and total weight were the highest in EG5 and the lowest in EG1. Gross fat content showed insignificant differences among the groups (p > 0.05). However, gross protein content and ash content showed significant differences across the groups (p < 0.05). The gross protein of the groups solely fed on artificial diets was lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. The contents of other amino acids, total amino acids (TAAs) and essential amino acids (EAAs) showed significant differences across the groups (p < 0.05). TAA, EAA and delicious amino acids of the groups fed solely on artificial diets were lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. Results indicated that the artificial diet (S2) can serve as substitutes of microalgal diets for P. martensii.     

Microalga <Emphasis Type="Italic">Isochrysis galbana</Emphasis> in feed for <Emphasis Type="Italic">Trachinotus ovatus</Emphasis>: effect on growth performance and fatty acid composition of fish fillet and liver

Seeking alternatives to the depleting fish oil are crucial for marine fish aquaculture, which is currently dependent on fish oil as the primary source of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs). Five isonitrogenous (46% crude protein) and isolipidic (16% crude lipid) feed diets (FO, ISO2.9, ISO4.8, ISO6.7, ISO8.6) were formulated by partially replacing fish oil with microalgae Isochrysis galbana. These diets were fed to triplicate tanks of Trachinotus ovatus (mean initial weight 1.92 g) for 80 days. This work demonstrates that a moderate inclusion (around 4.5–5.0 wt%, equivalent to the replacement of 24–26 wt% fish oil) of I. galbana biomass in fish diet improves fish growth performance, lipid deposition and enhances total n-3 fatty acids, DHA, and EPA contents in neutral and polar lipids (PLs) of fish muscle and liver of T. ovatus. The results disclosed in this study suggest that I. galbana microalgae represents a potential high-quality substitute for fish-based ingredients in aquaculture feeds, which can be a promising sustainable solution to resolve the depleting fish oil resource in a cost-effective manner.     

The effects of starvation on fast-start escape and constant acceleration swimming performance in rose bitterling (<Emphasis Type="Italic">Rhodeus ocellatus</Emphasis>) at two acclimation temperatures

To investigate the effects of starvation and acclimation temperature on the escape ability of juvenile rose bitterling (Rhodeus ocellatus), we measured the fast-start escape and constant acceleration swimming performance of fish fasted for 0 (control), 1 and 2 weeks and half-lethal periods (6 or 4 weeks) at two temperatures (15 and 25 °C). Fish acclimated at a high temperature exhibited shorter response latency (R), higher maximum linear velocity (V _max) and longer escape distance during escape movement (D _120ms) than those at the low temperature. Starvation resulted in a significant decrease in V _max and D _120ms at either low or high temperature and a significant increase in R at only the high temperature in the half-lethal period groups (P < 0.05). The relationship between V _max (Y, m s^?1) and starvation time (X, week) was Y ₁₅ = ?0.062X + 1.568 (r = ?0.665, n = 36, P < 0.001) at low temperature and Y ₂₅ = ?0.091X + 1.755 (r = ?0.391, n = 40, P = 0.013) at high temperature. The relationship between U _cat (Y, cm s^?1) and starvation time (X, week) was Y ₁₅ = ?1.649X + 55.418 (r = ?0.398, n = 34, P = 0.020) at low temperature and Y ₂₅ = ?4.917X + 62.916 (r = ?0.793, n = 33, P < 0.001) at high temperature. The slopes of equations showed a significant difference between low and high temperature (F _1,63 = 9.688, P = 0.003), which may be due to the different energy substrate utilization when faced with food deprivation at different temperatures.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Alternative microalgal diets for cultivation of the fairy shrimp <Emphasis Type="Italic">Branchinella thailandensis</Emphasis> (Branchiopoda: Anostraca)

Fairy shrimp is known as a nutritional food for fish and crustaceans in aquaculture. In most hatcheries, the microalga Chlorella sp. appears to be the most common, suitable, and nutritious food to feed fairy shrimp. In this study, we attempted to determine other alternative algal diets for cultivation of fairy shrimp Branchinella thailandensis. Seven experimental diets including three treatments of dried Spirulina sp. at 0.75 (S1), 1.5 (S2), and 3.0 mg dry weight individual^?1 (S3); three treatments of Chlorococcum humicola at 5 × 10⁵ (Ch1), 1 × 10⁶ (Ch2), and 2 × 10⁶ cells mL^?1 (Ch3); and a control diet (Chlorella vulgaris at 1 × 10⁶ cells mL^?1) were fed to 5-day-old shrimp for 15 days. Evaluation of growth performance, egg production, survival percentage, and nutritional and carotenoid content of the experimental fairy shrimp revealed that Ch3 is the most suitable algal diet. Our results suggest that C. humicola is the best alternative food source for the cultivation of B. thailandensis. In addition, dried Spirulina powder is also a good choice when live algae are not available and can be used as an alternative feed in fairy shrimp cultures.     

Effects of dietary <Emphasis Type="Italic">myo</Emphasis>-inositol on growth,chemical composition and plasma chemistry of Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis>

The present study was conducted to demonstrate the dietary myo-inositol requirement and its effects on the growth, proximate composition and blood chemistry of Amur sturgeon (Acipenser schrenckii). Triplicate groups of 30 fish (initial weight 11.90?±?0.12 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, 738.15 and 936.28 mg kg^?1) until satiation for 56 days. The fish were weighed after a 24-h fast, and six fish were used for whole body composition analysis. Further, the liver and muscle were sampled from another six fish for lipid analysis. The blood and liver were sampled from the remaining six fish for haematology and fatty acid analysis. The weight gain of fish increased with myo-inositol content, from the 28.75- to 343.83-mg kg^?1 myo-inositol treatment groups, and then stabilised. The liver lipid content and hepatosomatic index decreased significantly from 21.91 to 19.14% and from 3.20 to 2.76% with increased dietary myo-inositol supplementation, respectively. The whole body lipid content generally decreased from 6.33 to 5.55%. The content of liver-saturated fatty acids decreased significantly (28.13%) in the 936.28-mg kg^?1 treatment group. The content of plasma non-esterified fatty acids increased with the increase in dietary myo-inositol supplementation from 0.77 to 1.17 mmol L^?1, whereas the content of triglycerides significantly decreased from 4.62 to 3.28 mmol L^?1. In conclusion, the optimum myo-inositol requirement was found to be 336.1 mg kg^?1, based on weight gain in a two-slope quadratic broken-line model.     

Effects of dietary vitamin E supplementation on growth performance,fatty acid composition,lipid peroxidation and peroxisome proliferator-activated receptors (PPAR) expressions in juvenile blunt snout bream <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>

A 9-week feeding experiment was conducted to evaluate the effects of dietary vitamin E (VE) supplementation on growth performance, liver fatty acid composition, lipid peroxidation and peroxisome proliferator-activated receptors (PPAR) genes expressions in blunt snout bream juveniles. Fish (average initial weight: 0.59 g) were fed diet supplemented with 0, 50, 100, 300 and 500 mg α-tocopherol acetate/kg in triplicates, which were found to, respectively, contain 11.2, 56.3, 114.6, 306.5 and 588.4 mg α-tocopherol/kg diet. Results showed that final weight, body weight gain and specific growth rate significantly increased with increasing dietary VE supplemented level from 11.2 to 56.3 mg/kg. When the broken-line model was employed to estimate the adequate requirement of vitamin E based on body weight gain, the optimal level was 55.5 mg/kg in diet. Hepatosomatic index value significantly decreased with incremental dietary VE levels. However, liver VE concentration showed a direct relationship with the dietary VE level. The percentages of 20:5n-3, 22:6n-3 and total n-3 long chain polyunsaturated fatty acids in liver increased with increasing dietary VE supplementation. Meanwhile, the expressions of PPAR-α, PPAR-β and PPAR-γ in liver were down-regulated by supplementation of dietary VE level from 56.3 to 588.4 mg/kg. In conclusion, supplementation of more than 55.5 mg/kg vitamin E may improve growth and increase n-3 LC-PUFA content in blunt snout bream, which is beneficial to human consumer.     

Changes in the physicochemical and microbiological properties of dried anchovy <Emphasis Type="Italic">Engraulis japonicus</Emphasis> during storage

This study investigated the changes in the pH, thiobarbituric acid (TBA) value, volatile basic nitrogen (VBN), color L*, a*, b*, shear force, and microbial content (total plate count; TPC) of dried anchovy Engraulis japonicus in a home refrigerator and freezer at 2, ??1, ??5, and ??20 °C. The TBA value and shear force significantly decreased with increasing storage duration at all of the tested temperatures (p?<?0.05), whereas the VBN and TPC values increased (VBN significantly so) with increasing storage duration (p?<?0.05). The anchovy L* values decreased, whereas the a* (redness) and b* (yellowness) values increased as the storage period was lengthened. The pH decreased after 9 months. The results suggest that the optimum storage period of the dried anchovy is within 6 months. It can be stored below 2 °C for 3 months, but for storage exceeding 3 months, it should be frozen at ??5 °C.     

Growth and fatty acid composition of <Emphasis Type="Italic">Octopus vulgaris</Emphasis> paralarvae fed with enriched <Emphasis Type="Italic">Artemia</Emphasis> or co-fed with an inert diet

The rearing of Octopus vulgaris paralarvae during its planktonic life stage is a major challenge, as mortality is currently very high and unpredictable. In this study, we examined the survival and growth rates, as well as the fatty acid composition, of O. vulgaris paralarvae fed on three different dietary treatments: group ArDHA was offered juvenile Artemia enriched with a lipid emulsion (Easy DHA-Selco^®); group ArMA was fed with juvenile Artemia enriched with a mixture of microalgae (Rhodomonas lens and Isochrysis galbana); and group ArMA+ID received the same Artemia as group ArMA complemented with an inert diet. Dietary treatments were tested in triplicate with homogenous groups of paralarvae (25 individuals l^?1) established in 50-l tanks, and the experiment was conducted for 15 days. The survival rate of 15-day post-hatch (-dph) paralarvae from groups ArMA (20 ± 8%) and ArMA+ID (17 ± 4%) tended to be higher than in group ArDHA (13 ± 5%), though these differences were not statistically different. The dry weight (DW) of 15-dph paralarvae increased by almost 60% in groups ArMA and ArMA+ID, and nearly 40% in group ArDHA, with respect to hatchlings. The fatty acid (FA) composition of paralarvae revealed a remarkable drop of docosahexaenoic acid (22:6n-3, DHA) from hatchlings to 15-dph paralarvae of all groups (P < 0.05). However, paralarvae from group ArDHA contained higher levels of DHA than those from ArMA and ArMA+ID (P < 0.05). Despite Artemia enriched with DHA-Selco^® contained three-times more DHA than Artemia enriched with microalgae, no beneficial effects of this dietary treatment were observed on the performance of paralarvae.     

Growth performance and immune responses of gibel carp, <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>, fed with graded level of rare earth-chitosan chelate

A 12-week feeding trail was conducted to assess the effect of rare earth-chitosan chelate (RECC) on growth performance and immune responses of gibel carp, Carassius auratus gibelio. Isonitrogenous and isolipid experimental diets were supplemented with graded levels of rare earth-chitosan chelate (RECC 0, 0.8, 4 and 8 g Kg^?1). A total of 720 gibel carps (initial body weight about 14.32 g) were divided randomly into four groups with six replicates, respectively. Fish were fed with the experimental diets three times every day. At the end of the feeding trail, the survival rate was higher than 96 %. Weight gain rate and specific growth rate (SGR) significantly increased with RECC supplementation up to 0.8 g Kg^?1 (P < 0.05) and tended to decline at higher supplementation levels, while feed conversion ratio was not significantly different between groups (P > 0.05). Based on broken-line regression analysis of SGR, the optimum dietary RECC was estimated to be 0.71 g Kg^?1 of the diet. Condition factor and viscerosomatic index were not significantly affected by RECC (P > 0.05), while hepatosomatic index in the group fed with 0.8 g Kg^?1 RECC (3.45 ± 0.10 %) was significantly lower than in other groups (P < 0.05). Plasma ALT was significantly affected by RECC (P < 0.05), while AST was not. Plasma total protein and albumin were increased with RECC supplementation up to 0.8 g Kg^?1 and decreased significantly at higher supplementation levels (P < 0.05). RECC supplementation significantly decreased plasma urea and glucose concentration and increased plasma creatinine concentration significantly (P < 0.05). Respiratory burst activity of phagocytes and myeloperoxidase activity were not significantly different between groups, while superoxide dismutase activity and nitrogen monoxide concentration were increased with the increasing level of RECC in the diets. In conclusion, RECC could enhance growth performance and improve immunity of gibel carp.     

Transcripts of genes encoding reproductive neuroendocrine hormones and androgen receptor in the brain and testis of goldfish exposed to vinclozolin,flutamide, testosterone,and their combinations

Vinclozolin (VZ) is a pesticide that acts as an anti-androgen to impair reproduction in mammals. However, VZ-induced disruption of reproduction is largely unknown in fish. In the present study, we have established a combination exposure in which adult goldfish were exposed to VZ (30 and 100 μg/L), anti-androgen flutamide (Flu, 300 μg/L), and androgen testosterone (T, 1 μg/L) to better understand effects of VZ on reproductive endocrine system. mRNA levels of kisspeptin (kiss-1 and kiss-2) and its receptor (gpr54), salmon gonadotropin-releasing hormone (gnrh3) and androgen receptor (ar) in the mid-brain, and luteinizing hormone receptor (lhr) in the testis were analyzed and compared with those of control following 10 days of exposure. kiss-1 mRNA level was increased in goldfish exposed to 100 µg/L VZ and to Flu, while kiss-2 mRNA level was increased following exposure to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. gpr54 mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu and 100 µg/L VZ with T. gnrh3 mRNA level was increased in goldfish exposed to 100 µg/L VZ, to Flu, and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. The mid-brain ar mRNA level was increased in goldfish exposed to Flu and to combinations of 30 µg/L VZ with Flu, 100 µg/L VZ with T, and Flu with T. Testicular lhr mRNA level was increased in goldfish exposed to Flu and to combination of 30 µg/L VZ with Flu. These results suggest that VZ and Flu are capable of interfering with kisspeptin and GnRH systems to alter pituitary and testicular horonal functions in adult goldfish and the brain ar mediates VZ-induced disruption of androgen production.     

Haemato-biochemical,non-specific immunity,antioxidant capacity and histopathological changes in <Emphasis Type="Italic">Labeo rohita</Emphasis> fingerlings fed rubber protein isolate

A 60-day feeding trial was conducted to evaluate the haemato-biochemical, innate immune response, antioxidant capacity and histopathological changes in Labeo rohita fingerlings fed rubber protein isolates (RPI). One hundred and eighty fingerlings (average weight 4.45 ± 0.01 g) were distributed into five experimental groups in triplicate and fed with isonitrogenous and isocaloric diets. Soybean protein isolate (SPI) served as the reference diet (Control), and the treatment diets were formulated as RPI₂₅, RPI₅₀, RPI₇₅ and RPI₁₀₀ replacing 25, 50, 75 and 100% of SPI protein, respectively. The growth performance indices like final body weight (9.54–10.27 g), net weight gain (5.09–5.84 g), metabolic growth rate (4.54–5.02) and feed efficiency ratio (0.60–0.65) among the various groups were not significantly different (P > 0.05). All the haematological parameters, except red blood cells, showed no significant differences compared with the control group (P > 0.05). The immuno-biochemical parameters like albumin, globulin, total immunoglobulin, respiratory burst and lysozyme activities among the various groups did not differ significantly (P > 0.05). The stress enzyme such as superoxide dismutase (SOD), catalase (CAT) and oxidative stress marker malondialdehyde (MDA) showed no significant difference (P > 0.05). Histopathological examination of the liver revealed no marked changes. In summary, the results showed that RPI was well utilised by the fish and its inclusion did not generate any oxidative-induced stress, thus, RPI may be suggested as a potential replacement for SPI in fish diets without any detrimental effects. Hence, protein isolation offers a unique opportunity for the utilisation of rubber seed meal.     

Genetic polymorphisms and DNA methylation in exon 1 CpG-rich regions of <Emphasis Type="Italic">PACAP</Emphasis> gene and its effect on mRNA expression and growth traits in half smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The pituitary adenylate cyclase activating polypeptide (PACAP) is a new type of hypophysiotropic hormone and plays an important role in regulating the synthesis and secretion of growth hormone and gonadotropin. The research on the relationship between PACAP and different growth traits would contribute to explain its function during the process of growth. Moreover, epigenetic modifications, especially DNA methylation at the CpG sites of the SNPs, play important roles in regulating gene expression. The results suggest that a SNP mutation (c.C151G) in the PACAP gene of male half smooth tongue sole (Cynoglossus semilaevis) is significantly associated with growth traits and serum physiological and biochemical parameters such as inorganic phosphorus (P < 0.05). The SNP is located in a CpG-rich region of exon 1. Intriguingly, the transition (C→G) added a new methylation site of PACAP gene. This SNP was also significantly related to the expression and methylation level of PACAP (P < 0.05). Individuals with GG genotype had faster growth rates than those of CG and CC genotypes. Moreover, GG genotype had significantly higher PACAP expression level and lower methylation level than CG and CC genotypes. In the serum indexes, only inorganic phosphorus content within GG genotypes was significantly higher than CC genotypes. This implied that the mutation and methylation status of PACAP gene could influence growth traits and this locus could be considered as a candidate genetic or epigenetic marker for Cynoglossus semilaevis molecular breeding.     

Quantitative analysis of <Emphasis Type="Italic">Ichthyobodo salmonis</Emphasis> an ectoparasitic flagellate infecting juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> in hatcheries

Ichthyobodosis caused by the ectoparasitic flagellate Ichthyobodo salmonis is a significant cause of mortality in juvenile chum salmon Oncorhynchus keta reared in hatcheries of northern Japan. The present study established a real-time quantitative polymerase chain reaction assay (qPCR) of I. salmonis ribosomal DNA (rDNA) using SYBR Green. This assay allows monitoring of parasite infections for the epidemiological study and control of ichthyobodosis in hatcheries. qPCR showed high reproducibility for measurements between 1.0 and 1.0 × 10⁸ rDNA copy/μl. There was a significant positive correlation between the number of parasites and the amount of I. salmonis rDNA. A survey using the qPCR assay indicated that infection by I. salmonis was present in 23 of 87 hatcheries; parasite loads were estimated to be between 50 and 750 parasites/g juvenile body weight. These results demonstrate that our qPCR assay enables the surveying of juvenile chum salmon reared in hatcheries for infection by I. salmonis.     

Long-term effects of stocking density on survival,growth performance and marketable production of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

Sea urchins were stocked at a density of 15 (D15), 30 (D30), 45 (D45) and 60 (D60) urchins/cage (0.3 m long × 0.2 m wide × 0.4 m high) in a laboratory culture environment for 16 months. The wet body weight (BW) and test diameter growth were monitored at 2-month intervals during the experiment. At the conclusion of the experiment, the surviving sea urchins were counted and the gonad wet weight (GW) and gonad color were measured. Specific growth rate (SGR) of body weight, survival rate (SR), gonad index (GI), gonad color difference (ΔE ₀₀), coefficients of variation (CV) of BW, GW, GI and ΔE ₀₀, total biomass yield (TY) and total gonad yield (TGY) per cage were calculated. Two marketable yield variables, graded according to gonad index, i.e., marketable biomass yield (MY) and marketable gonad yield (MGY), were also calculated. Coefficient of variation of final body weight (FW) and final test diameter (FTD) of sea urchins increased as the stocking density increased, indicating the existence of adverse social interactions. These adverse social interactions detrimentally affected FW, FTD, SGR, GW and GI (P < 0.01). Although SR decreased with the increasing densities, no statistical significant difference was detected. Sea urchins at D15 had the lowest gonad color difference (ΔE ₀₀) (P < 0.05). However, statistically equal CV of ΔE ₀₀ indicates this density effect was not a result of adverse social interactions. TY and TGY increased with increased density and can be described by the following equations: TY = 84.18X ^0.64, R ² = 0.999 and TGY = 24.16X ^0.38, R ² = 0.979. However, the MY and MGY were not significantly different among stocking densities. The results of this study demonstrate that in intensive culture S. intermedius at low stocking density can achieve high growth rate, gonad index and desirable color without decreasing the marketable yield. Farmers should choose to culture S. intermedius at low stocking densities.