Preliminary Study of the Dietary α‐Tocopherol Requirement in Sea Cucumber,Apostichopus japonicus

This study was conducted to evaluate the dietary α‐tocopherol (vitamin E) requirement in juvenile sea cucumber, Apostichopus japonicus. Sea cucumbers averaging 1.48 ± 0.07 g (mean ± SD) were randomly distributed into 18 rectangular plastic tanks of 20 L capacity in a recirculating system (20 animals per tank). Six semi‐purified experimental diets with average protein and crude lipid levels (dry matter) of 29.7 ± 0.36% and 4.39 ± 0.23% (mean ± SD), respectively were formulated to contain 0 (E4), 15 (E12), 30 (E23), 60 (E44), 120 (E77) and 600 (E378) mg α‐tocopherol/kg diet, supplied as dl‐α‐tocopheryl acetate. Diets were analyzed for α‐tocopherol content by HPLC and the α‐tocopherol levels were 4.01, 12.4, 23.1, 44.3, 77.4 and 378 mg α‐tocopherol/kg diet for E4, E12, E23, E44, E77 and E378 diets, respectively. Casein and defatted fish meal were used as the protein sources in the diets while wheat flour was the carbohydrate source. Sea cucumbers were fed each of the six experimental diets in triplicate groups. At the end of the 14‐week feeding trial, weight gain (WG), specific growth rate (SGR) and feed efficiency (FE) of sea cucumbers fed on E23, E44, E77 and E378 diets were significantly (P < 0.05) higher than those of animals fed on E4 and E12 diets. However, there were no significant differences in WG, SGR and FE among sea cucumbers fed on E23, E44, E77 and E378 diets or among those fed on E4 and E12 diets. Survival of sea cucumbers fed on E44, E77 and E378 diets were significantly higher than those of animals fed on E4, E12 and E23 diets. However, there were no significant differences among sea cucumbers fed on E4, E12 and E23 diets or among those fed on E44 and E77 diets. Whole‐body vitamin E concentration increased with α‐tocopherol content of the diets. Broken line analysis of WG showed an optimum dietary α‐tocopherol requirement of 41 mg α‐tocopherol/kg diet in sea cucumber. These results indicated that the optimum dietary α‐tocopherol requirement in sea cucumber in the form of dl‐α‐tocopheryl acetate could be higher than 23.1 mg α‐tocopherol/kg diet but lower than 44 mg α‐tocopherol/kg diet.     

Daily and Annual Variation in Digestive Enzymes – Amylase and Basic and Acid Proteases – in Gilt‐head Sea Bream,Sparus aurata

This study examines the daily and annual changes in amylase and basic and acid protease activities in gilt‐head sea bream, Sparus aurata. During the experiment, the fish were kept in cages for production under natural temperature and photoperiod conditions and fed with a commercial diet. To study the effect of photoperiod and temperature, the experiments were conducted during six different months that were chosen based on temperature and photoperiod. To determine daily changes in enzymes activities, for every 3 h during 24 h period, six fish were randomly selected and sacrificed. The annual results for acid protease activity show a rhythm with 6‐mo period (bimodal rhythm) with acrophases in May and November. The activities of pancreatic enzymes, which include basic protease and amylase, exhibited parallel changes with two peaks of activity in January and October without a rhythmic pattern. The daily changes in enzyme activities are significant only in May, June, and November for basic proteases and May, October, and November for amylase (P < 0.05). The observed variations suggest a varying capacity for digestion depending on the day and the year and could aid in the study of seasonal feeding protocols.     

Gene Cloning and Expression of Major Capsid Protein of Large Yellow Croaker Iridovirus and Its Secretion Based on α‐hemolysin Transport System

Large yellow croaker iridovirus (LYCIV) is an important pathogen of mariculture fish. The complete gene of an important protective antigen, major capsid protein (MCP), of LYCIV was amplified by polymerase chain reaction and was cloned into pET‐16b for expression in Escherichia coli. The MCP‐encoded gene was in‐frame fused to the E. coliα‐hemolysin transport elements to construct the MCP secretory expression plasmid pMOhlyM, and its secretion characters in E. coli and attenuated Vibrio anguillarum MVAV6203 were investigated. With the aid of E. coliα‐hemolysin transport system, about 400 μg/L MCP was secreted into culture supernatant in E. coli, while intracellularly expressed MCP in MVAV6203 was not secreted. The study provides the foundation for attempts to secrete viral‐originated antigens by the E. coli HlyA transporter in bacterial hosts and to explore feasible ways to develop multivalent live vaccines against both bacterial and viral pathogens.     

Antiparasitic activity of the antimicrobial peptide HbβP‐1, a member of the β‐haemoglobin peptide family

A family of antimicrobial peptides (AMPs) derived from the β‐subunit of haemoglobin was recently isolated from channel catfish, Ictalurus punctatus, infected with Ichthyophthirius multifiliis (ich), an important freshwater fish parasite that causes ichthyophthiriosis. We previously discovered that one of these AMPs, HbβP‐1, had strong cidal activity against ich as well as another ectoparasite, Tetrahymena pyriformis. HbβP‐1 toxicity was specific, primarily affecting the trophozoite (trophont) stage of ich. Here, we show that HbβP‐1 acts more rapidly to kill smaller (presumably less mature) trophonts of ich, taking almost twice as long to kill larger trophonts (P < 0.0001). It acts more rapidly than an unrelated AMP, piscidin 1, which is haemolytic and also lethal to ich trophonts. HbβP‐1 is potently and selectively lethal to the trophont stage of the dinoflagellate ectoparasite, Amyloodinium ocellatum, one of the most important pathogens of warmwater marine fish. HbβP‐1 has no effect on the fish gill cell line feeder layer (G1B cells) used to propagate Amyloodinium, further suggesting a highly selective action. These findings suggest that HbβP‐1 or related AMPs might function in protecting marine as well as freshwater fish and that HbβP‐1 has highly selective activity against specific life stages of important fish ectoparasites.     

The primary culture and subculture of lymphoid cells from shrimp,Penaeus chinensis on thermo‐sensitive CS/α, β‐GP hydrogel

A thermo‐sensitive hydrogel composed of chitosan and α, β‐glycerophosphate was successfully synthesized and used as basic substrate for primary culture and subculture of lymphoid cells from shrimp, Penaeus chinensis. When the ambient temperature increases from 4°C to 25°C, the chitosan/α, β‐glycerophosphate solution transforms into a semi‐solid three‐dimensional hydrogel with a rough surface. Primary lymphoid cells formed were round and grew homogenously over the surface of the hydrogel. The peroxidase activity indicated that cells seeded on the hydrogel suffered less stress than cells suspended in control culture flasks. The higher relative viability and alkaline phosphatase activity of primary cultured lymphoid cells indicated that the chitosan/α, β‐glycerophosphate hydrogel enhanced the growth and proliferation of lymphoid cells. Subsequent subculture of lymphoid cells starting from crumbled primary hydrogel culture was also successful.     

A3α‐peptidoglycan extracted from Bifidobacterium sp. could enhance immunological ability of Apostichopus japonicus

To assess the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the immune response and disease resistance of sea cucumber, different concentrations (0, 0.5, 5 and 50 mg mL^?1) of A3α‐PG suspensions were used to perform hypodermic injection on Apostichopus japonicus, followed by a Vibrio splendidus challenge. Total coelomocyte count (TCC), phagocytosis activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extra‐cellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD) and peroxidase (POD), were measured at 2, 6, 14 and 24 h post injection (hpi). The TCC was not significantly affected (P > 0.05) by A3α‐PG, ranging from 1.84 × 10⁶ to 3.53 × 10⁶ cells mL^?1. The coelomocyte phagocytosis activity was significantly activated (P < 0.05) in all the A3α‐PG treatments, whereas no significant difference was observed between them except 24 hpi (P > 0.05). The EC‐ACP activity in the 5.0 mg mL^?1 treatment increased significantly (P < 0.05) at all sampling times, while the IC‐ACP activity in the 50 mg mL^?1 treatment increased significantly (P < 0.05) at 2 hpi. Also, the 5.0 mg mL^?1 treatment had significant (P < 0.05) increase in the EC‐ALP activity within 14 hpi and the EC‐POD activity at 2 hpi, respectively, while significantly (P < 0.05) enhanced IC‐ALP and IC‐POD activities were observed in the 50 mg mL^?1 treatment within 6 hpi and at 2 hpi, respectively. Only the 5.0 mg mL^?1 treatment showed significant (P < 0.05) increase in the EC‐SOD activity at 2 hpi and IC‐SOD activity within 14 hpi, respectively. The challenge test showed that the animals treated with 50 mg mL^?1 of A3α‐PG had notably lower cumulative mortality after 14 days following V. splendidus exposure. All together, these results suggest that A3α‐PG could positively enhance immune response that effectively promotes the health status of A. japonicus against V. splendidus infection.     

Immunostimulatory effects of dietary poly‐β‐hydroxybutyrate in European sea bass postlarvae

The stable production of high‐quality fry in marine aquaculture is still hampered by unpredictable mortality caused by infectious diseases during larval rearing. Consequently, the development of new biocontrol agents is crucial for a viable aquaculture industry. The bacterial energy storage compound poly‐β‐hydroxybutyrate (PHB) has been shown to exhibit beneficial properties on aquatic organisms such as enhanced survival, growth, disease resistance and a controlling effect on the gastrointestinal microbiota. However, the effect of PHB on the developing immune system of fish larvae has so far not been investigated. In this study, the effect of feeding PHB‐enriched Artemia nauplii on survival, growth and immune response in European sea bass (Dicentrarchus labrax) postlarvae was examined. Amorphous PHB was administered to 28‐day‐old sea bass postlarvae over a period of 10 days. The survival and growth performance were monitored, and the expression of 29 genes involved in immunity, growth, metabolism and stress‐response was measured. While the expression of the insulin‐like growth factor 1 (igf1), an indicator of relative growth, was upregulated in response to feeding PHB, the larval survival and growth performance remained unaffected. After 10 days of PHB treatment, the expression of the antimicrobial peptides dicentracin (dic) and hepcidin (hep) as well as mhc class IIa and mhc class IIb was elevated in the PHB fed postlarvae. This indicates that PHB is capable of stimulating the immune system of fish early life stages, which may be the cause of the increased resistance to diseases and robustness observed in previous studies.     

Effect of Different Feeding Levels of Plant‐ingredient‐based Feed on Fillet Fatty Acid Profile,Carcass Trait,and Sensory Characteristics of Indian Major Carps in Earthen Pond Polyculture

The objective of the present study was to evaluate the effect of plant‐ingredient‐based feeds on fillet fatty acid profiles, proximate composition, carcass traits, and sensory characteristics of three Indian major carps (IMCs), Catla catla, Labeo rohita, and Cirrhinus mrigala, when fed at different levels of their body weight. An experimental feed was prepared with only plant ingredients. The IMCs were fed twice daily at 1.0, 1.5, 2.0, and 2.5% of their body weight for 150 d in 0.06‐ha earthen pond polyculture systems. The maximum growth and best feed utilization was achieved with feeding at 2% of fish biomass in the ponds. Fillet yield and lipid recovery increased significantly (P < 0.05), with increasing feeding level up to 2% of body weight and plateaued thereafter. In fillets, eicosapentaenoic acid (20:5n‐3) and docosahexaenoic acid (22:6n‐3) concentration increased with increasing feeding levels. Carcass traits, sensory characteristics, and consumer acceptance of IMC fillets were not influenced by feeding plant‐ingredient‐based feeds at different feeding rates.     

Comparative effect of Freund's adjuvant and Aloe vera L. gel on the expression of TNF‐α and IL‐1β in vaccinated Cyprinus carpio L. with Aeromonas hydrophila C. bacterin

The comparative effects of Freund's and Aloe vera gel as adjuvants on the expression of IL‐1β and TNF‐α genes were studied in vaccinated common carp (Cyprinus carpio) with Aeromonas hydrophila bacterin. Fishes were intraperitoneally immunized with A. hydrophila bacterin in combination with Aloe vera gel or Freund's and also without any adjuvant. At day 28 after immunization, all groups were challenged by lethal dose of A. hydrophila (10⁷ cells/fish). Changes in the expression of IL‐1β and TNF‐α genes were evaluated in anterior kidney before challenge and 12, 24, 72 and 7 days postchallenge using quantitative real‐time PCR. Higher expression levels of both genes were observed in all vaccinated groups compared with non‐immunized group. Fishes which received Aloe vera gel showed higher expression of IL‐1β and TNF‐α in relation to animals which vaccinated with or without Freund's adjuvant. We concluded that Aloe vera gel in compared with Freund's adjuvant had a more stimulatory effect on the expression of immune‐related genes in vaccinated common carp and it can use as a novel adjuvant in aquaculture.     

The effects of different proportions of the 17β‐estradiol and 17α‐methyltestosterone on growth,sex reversal and skin colouration of the electric blue hap (Sciaenochromis ahli Trewavas, 1935)

This study was performed to investigate the effects of 17β‐estradiol (ES) and 17α‐methyltestosterone (MT) on growth, development, survival, sex ratio and colour change in the electric blue hap (Sciaenochromis ahli Trewavas, 1935). The hormones were not supplemented to the control feed, while six other feeds were prepared by adding 20, 40 and 60 mg kg^?1 17β‐ES or 20, 40 and 60 mg kg^?1 17α‐MT to each, resulting in seven different feed treatments. Average live weight of the fish supplemented with these diets was 0.42 ± 0.04 g. At the end of the study, the highest weight gain was observed in fish fed 60 mg kg^?1 17α‐MT group (2.62 ± 0.11 g) and the difference with the groups fed with 17β‐ES was found to be significant. All fish fed 17α‐MT were male, while the rates of feminization in fish fed 17β‐ES at 20, 40, 60 mg kg^?1 were 91.11%, 88.88% and 93.33% respectively. Survival rates were respectively determined as 80%, 95.56%, 84.44%, 93.33%, 77.78%, 84.44% and 84.44% for the control, 20, 40, 60 mg kg^?1 17β‐ES and 20, 40, 60 mg kg^?1 17α‐MT treatments. The best colouration was achieved in the 17α‐MT groups (P < 0.05). The L* values varied between 32.98 ± 4.44 and 61.35 ± 2.19, a* values between ?7.06 ± 0.22 and ?3.42 ± 0.11, and b* values between ?7.74 ± 0.10 and 11.65 ± 0.03, while Chroma (C*) and Hue (H°_ab) angle values varied between 7.54 ± 0.22 and 13.60 ± 0.01 and between 119.76 ± 0.05 and 239.73 ± 4.86. In conclusion, the 17α‐MT feeding was found to have a greater effect on the growth, feed conversion ratio, masculunization and pigmentation of the electric blue haps than the 17β‐ES treatment.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Effects of anthropogenic disturbances on α and β diversity of fish assemblages and their longitudinal patterns in subtropical streams,China

Identifying the spatial patterns of α and β diversity of biotics is an important yet little‐understood area of basic and applied ecological research. Although the upstream–downstream patterns of α diversity of stream fishes are numerously reported, β diversity has received less attention. In this study, we surveyed fishes along the upstream–downstream gradients in three headwater streams of the Qingyi River, China, which were affected by different extents of human activities. We aimed to assess how anthropogenic disturbances affect α and β diversity of stream fishes and their upstream–downstream patterns. We found that, compared with that in the Shuxi Stream disturbed less, endemic species decreased and cosmopolitan species increased in the Maxi and Puxi Stream disturbed heavily. The streams disturbed heavily showed lower α diversity and higher β diversity than that in the stream disturbed less. This among‐stream variations in fish diversity only occurred at the mid‐downstream, not upstream segments. α diversity increased downstream and β diversity decreased downstream from headwaters to mouth in the stream disturbed less, whereas this upstream–downstream pattern in fish diversity shifted in the streams disturbed heavily, in which both α and β diversity showed the quadratic distributions with the hump‐shape for α diversity and the U‐shape for β diversity respectively. Our results suggest that anthropogenic disturbances cause some cosmopolitan fishes replacing many endemic fishes in upland streams. This replacement processes result in α diversity decreasing and β diversity increasing, and shift the spatial patterns in fish diversity along the upstream–downstream gradient.     

Effects of dietary supplementation of A3α‐peptidoglycan on the growth,immune response and defence of sea cucumber Apostichopus japonicus

To determine the effects of A3α‐peptidoglycan (A3α‐PG) extracted from Bifidobacterium sp. on the growth, immune response and disease resistance of sea cucumber Apostichopus japonicus, a 70‐day feeding trial was conducted in this study. A total of 216 sea cucumbers were fed with four practical diets prepared from a commercial feed with different contents (0, 1.5, 2.5 and 4.0 g kg^?1) of A3α‐PG. The specific growth rate (SGR), total coelomocyte count (TCC), phagocytotic activity and activities of four immunological enzymes in both cell‐free coelomic fluid (extracellular, EC) and coelomocyte lysate supernatant (intracellular, IC), including acid phosphatase (ACP), alkaline phosphatase (ALP), peroxidase (POD) and superoxide dismutase (SOD), were measured at the end of the feeding trial. Finally, the animals were administered a 16‐day Vibrio splendidus challenge via intraperitoneal injection to test the potency of A3α‐PG on disease resistance. Compared with the control (0 g kg^?1 A3α‐PG), a significant increase (P < 0.05) in SGR was observed in the groups fed with 1.5 and 2.5 g kg^?1 A3a‐PG. The TCC, ranging from 7.25 × 10⁶ to 1.05 × 10⁷ cells mL^?1, was not significantly affected (P > 0.05) by A3α‐PG,. Coelomocyte phagocytotic activities in all of the A3α‐PG‐supplemented groups were significantly activated (P < 0.05), but no significant difference (P > 0.05) was observed. Sea cucumbers fed with 1.5 and 2.5 g kg^?1 A3α‐PG exhibited significant activation (P < 0.05) of EC/IC‐ACP, EC/IC‐ALP, and EC/IC‐POD activities. A significant increase in EC‐SOD activities (P < 0.05) was exhibited by all groups with A3α‐PG supplementation. The challenge test showed that animals fed with diets containing 2.5 and 4.0 g kg^?1 A3α‐PG had significantly lower cumulative mortalities compared with the control 16 days after exposure. All of the results presented here show that A3α‐PG can positively enhance the growth, immune response and disease resistance of sea cucumber, suggesting that dietary supplementation of A3α‐PG has potential applications in the health management of economic species of sea cucumber.     

LHRHa‐induced ovulation of the endangered‐Caspian brown trout (Salmo trutta caspius) and its effect on egg quality and two sex steroids: testosterone and 17α‐hydroxyprogestrone

To induce synchronized ovulation, migrating wild Caspian brown trout (Salmo trutta caspius) females were treated with two interperitoneal injections of Des‐Gly¹⁰, d ‐Ala⁶ LHRH (LHRHa), given 3 days apart. Two injections of 100 μg kg^?1 body weight of this hormone effectively induced ovulation. Within 27 days from the second injection, all fish injected with 100 μg kg^?1 LHRHa had ovulated compared with 54.5% of the controls. The mean time to ovulation was reduced significantly (P<0.05) from 31.67±4.84 days in control fish and 28.83±7.31 days in sham‐treated fish to 16.36±1.61 days in fish injected with 100 μg kg^?1 LHRHa. The fertilization rate in 50 and 100 μg kg^?1 LHRHa‐injected fish was significantly lower than that in the control fish (P<0.05). In fish injected with 50 and 100 μg kg^?1 LHRHa, significant (P<0.05) changes in testosterone (T) and 17α‐hydroxyprogestrone (OHP) levels were observed. After the second LHRHa injection, the fish injected with 100 μg kg^?1 showed the highest serum levels of testosterone and OHP. These results demonstrate that the use of LHRHa can effectively reduce the mean time to ovulation and induce synchronized ovulation in Caspian brown trout.     

Dietary α‐Ketoglutarate supplementation alleviates harmful effects of high environmental ammonia on grass carp,Ctenopharyngodon idella

Current intensive fish farming usually causes high environmental ammonia (HEA) in ponds that is toxic to fish. α‐Ketoglutarate (α‐KG) can be rapidly transaminated to glutamic acid and further aminated to glutamine. Therefore, we hypothesized that dietary α‐KG supplementation would alleviate HEA toxicity to fish. To test the hypothesis, 270 healthy grass carp (Ctenopharyngodon idella) juveniles were randomly assigned to control, HEA (18.37 mg/L ammonia) and HEA + α‐KG (0.75% of α‐KG) groups. Ammonia and free amino acid content in plasma and brain, liver glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activity, and urea and glycogen content were measured on the first, seventh and 42nd days. Our results showed short‐term HEA exposure (1 day) led to a significant ammonia accumulation in the brain and plasma and significantly decreased glutamic and aspartic acid content in the brain and increased glutamine content in the brain and plasma. The long‐term HEA exposure (42 days) caused significant reductions in glycine and arginine content in the brain tissue. In most cases, dietary α‐KG supplementation alleviated the fluctuations in FAA content in the brain and plasma. Our results suggested dietary α‐KG alleviated HEA toxicity to grass carp.     

Influence of dietary linoleic acid (18:2n‐6) and α‐linolenic acid (18:3n‐3) ratio on fatty acid composition of different tissues in freshwater fish Songpu mirror carp,Cyprinus Carpio

Six isonitrogenous and isoenergetic purified diets were formulated to feed Songpu mirror carp for 60 days. The control diet (CD) was only supplemented with soybean oil. The other five experimental diets contained soybean oil, linseed oil and lard oil blended at various inclusion levels to attain different linoleic acid (LA)/α‐linolenic acid (LNA) ratios (0.53, 1.04, 2.09, 3.95, 6.82) with a constant total C₁₈ polyunsaturated fatty acids (PUFA; LA+LNA, 2% dry weight) content. The fatty acid (FA) profiles of hepatopancreas, dorsal muscle, intestine, intraperitoneal fat (IPF), spleen and kidney reflected those of the diets, but with some differences. The spleen showed the lowest correlation with diet compared with other tissues, followed by the hepatopancreas (P < 0.05). The intestine and IPF showed relatively higher correlation. On the other hand, the control group had the lowest tissue‐diet correlation, followed by the LA/LNA0.53 group (P < 0.05), whereas the LA/LNA2.09 showed the highest. The LA/LNA ratios in the tissues were up‐regulated in the LA/LNA0.53, 1.04 groups and down‐regulated in the LA/LNA3.95, 6.82 groups. This was due to when LA (or LNA) was highly added in diet, the decrease in this FA was huge in tissue. The contents of saturated fatty acids and monounsaturated fatty acids increased in the control group, but seemed not influenced by dietary LA/LNA ratios. These results demonstrated that the FA deposition was tissue‐specific, and also influenced by the dietary FA composition in the experimental fish. Finally, we suggest that 2.09 is the optimal LA/LNA ratio (2% C₁₈ PUFA) of Songpu mirror carp for fillet FA composition.