Enhancement of Hsp70 synthesis protects common carp, Cyprinus carpio L., against lethal ammonia toxicity

Exposure to TEX‐OE^®, a patented extract of the prickly pear cactus (Opuntia ficus indica) containing chaperone‐stimulating factor, was shown to protect common carp, Cyprinus carpio L., fingerlings against acute ammonia stress. Survival was enhanced twofold from 50% to 95% after exposure to 5.92 mg L^?1 NH₃, a level determined in the ammonia challenge bioassay as the 1‐h LD50 concentration for this species. Survival of TEX‐OE^®‐pre‐exposed fish was enhanced by 20% over non‐exposed controls during lethal ammonia challenge (14.21 mg L^?1 NH₃). Increase in the levels of gill and muscle Hsp70 was evident in TEX‐OE^®‐pre‐exposed fish but not in the unexposed controls, indicating that application of TEX‐OE^® accelerated carp endogenous Hsp70 synthesis during ammonia perturbation. Protection against ammonia was correlated with Hsp70 accretion.     

Anaemia and plasma lipid profile in common carp (Cyprinus carpio) exposed to ambient copper sulphate and nano‐scale copper oxide

The objective of this study was to determine the effects of copper sulphate (CS) and copper oxide nanoparticles (NCuO) on haematological characteristics and plasma lipid profile in Cyprinus carpio. Fish were exposed to CS (0.25 mg L^?1 Cu) and NCuO [0.25 (LNP) and 25 mg L^?1 (HNP) Cu] for 14 days. CS and LNP treatments showed hypochromic anaemia after 3 and 7 days, and macrocytosis and haemolytic anaemia after 14 days. HNP treatment showed hypochromic anaemia and polycythemia after 3 days, and macrocytosis and hypochromic anaemia after 7 days and haemolytic hypochromic anaemia after 14 days. All copper‐exposed fish showed leukopenia at all times, except for HNP after 7 days and CS after 3 days. CS treatment at all times, and NCuO treatments after 14 days showed elevated plasma bilirubin. CS (3rd and 7th days) and HNP (3rd day) treatments had significantly higher plasma triglyceride compared to the control. Significant increases in plasma cholesterol and lipoprotein levels were observed after 3 and 14 days in CS, and after 14 days in LNP treatment; whereas HNP treatment had significantly higher high density lipoprotein (HDL) and very low‐density lipoprotein (VLDL) levels after 3 days, and cholesterol, HDL and low‐density lipoprotein (LDL) after 14 days. In conclusion, CS and NCuO induce anaemia probably due to iron metabolism disorder and RBC destruction in carp. CS and NCuO (at high concentration) alter plasma lipid profile, which may be due to stress‐induced lipolysis and hepatobiliary damages. At similar concentrations, copper nanoparticles are less toxic to carp than copper ions.     

Molecular cloning and expression of kidney‐type glutaminase from common carp (Cyprinus carpio) and its up‐regulation by glutamine in primary culture enterocyte

Glutaminase (GLS) is the key enzyme of glutamine (Gln) metabolism and utilization. In this study, a cDNA encoding GLS protein was identified from common carp Cyprinus carpio intestine. The open reading frame of GLS cDNA encodes a polypeptide of 595 amino acids, which shows a high similarity with its zebrafish Danio rerio counterpart. Bioinformatic analysis showed the protein belongs to kidney‐type GLS. The putative protein has glutaminase domain and ankyrin repeats domain, which are highly conserved among vertebrate orthologues. Real‐time quantitative PCR analysis revealed that the abundance of GLS mRNA was the highest in the white muscle, followed by the brain, eyeball and pituitary. Glutaminase was ubiquitously expressed in all intestinal segments of common carp. The activity of GLS did not distribute uniformly along the entire length of the intestine. In primary culture enterocyte, and the expression of GLS mRNA is up‐regulated quickly and effectively by Gln.     

Antibacterial efficacy and pharmacokinetic evaluation of sanguinarine in common carp (Cyprinus carpio) following a single intraperitoneal administration

Sanguinarine (SA), with antimicrobial and antiparasitic activities against fish pathogens, exhibits great potential commercial use in aquaculture. However, little information on pharmacokinetics of SA restricts further application in aquaculture. In this study, pharmacokinetics of SA in common carp (Cyprinus carpio) following a single intraperitoneal administration [10 mg kg^?1 BW (body weight)] was evaluated by high‐performance liquid chromatography (HPLC). The peak concentration (C_max) of SA in kidney was 11.8 μg g^?1, which was higher than in other tissues and plasma. The terminal half‐life in fish tissue and plasma was as follows: 42.3 h (kidney) > 37.2 h (liver) > 20.1 h (gill) > 18.8 h (muscle) > 10.9 h (spleen) > 10.0 h (plasma). Additionally, we determined the bacterial loads in tissues of common carp infected with Aeromonas hydrophila after i.p. administration of SA at 0, 5, 10 and 20 mg kg^?1 BW. The results showed that i.p. administration of SA at 10 mg kg^?1 BW significantly enhanced antibacterial efficacy against A. hydrophila, where the antibacterial ratio in the gill, kidney, spleen and liver on day 5 was 95.13%, 93.33%, 90.09% and 92.82%, respectively. Overall, these results suggested the potential of SA to treat A. hydrophila infection in common carp farming industry.     

Effects of temperature and dietary protein on gene expression of Hsp70 and Wap65 and immunity of juvenile mirror carp (Cyprinus carpio)

Juvenile mirror carp were fed diets containing 303.4, 321.7, 341.2, 361.0 and 379.1 g kg^?1 proteins, respectively, and reared at different water temperatures (18, 23 and 28°C) for 60 days. Gene expression of heat shock protein gene (Hsp70) and the warm temperature acclimation‐related 65 kDa protein gene (Wap65), immunity and antioxidant status in the carp were investigated. Results indicated that the contents of serum complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM), as well as activities of liver superoxide dismutase (SOD) and lysozyme (LSZ) were significantly enhanced with increasing dietary protein (P < 0.05), while content of malondiadehyde (MDA) decreased. Gene expression level of Wap65 in the liver significantly increased with dietary protein, while gene expression of Hsp70 decreased. The contents of C3, C4 and IgM, the activities of SOD and LSZ and gene expression level of Wap65 in the liver significantly increased with temperature. These results suggest that: Serum immune parameter, antioxidant enzymes and Hsp70 and Wap65 expression interact in fish to improve ability to adapt to the environment; and the optimal conditions for the immunity of carp are 348.1?354.5 g kg^?1 protein at 18°C, 352.3?364.9 g kg^?1 at 23°C and 360.2?364.3 g kg^?1 at 28°C, and the optimum temperature for carp is 23°C.     

The effects of dietary inulin on growth performances,survival and digestive enzyme activities of common carp (Cyprinus carpio) fry

This study was conducted to investigate the effects of dietary inulin on growth performance, diet utilization, survival rate, carcass composition and digestive enzymes activities (amylase, lipase and protease) of carp (Cyprinus carpio) fry (0.55 ± 0.02 g). After acclimation, fish were allocated into 9 tanks (40 fish per tank) and triplicate fish groups were fed, control diet (0 g) or diets containing 5 g and 10 g inulin kg^?1 for 7 weeks. No significant effect on growth performance and diet utilization of fish fed inulin compared with the control group was observed. However, supplementation of inulin significantly increased survival rate and carcass lipid content, while carcass protein content significantly decreased. Dietary inulin had no significant effects on digestive lipase, protease and amylase activities.     

热应激对大正三色锦鲤非特异性免疫指标及HSP70基因表达的影响

为探讨锦鲤的抗逆机理,在实验室条件下研究了持续热应激对大正三色锦鲤非特异性免疫指标及HSP70基因相对表达量的影响。分别于应激前、应激后2、6、10、14、18、22、26 h进行取样测定呼吸爆发、补体蛋白3(C3)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及热休克蛋白70(HSP70)基因的相对表达量。结果发现,热应激后呼吸爆发降低,在应激后2、10、18～26h降低显著(P<0.05);热应激2 h后血清中C3含量略有上升,应激6～26 h过程中与应激前相比均下降,应激14～22 h显著地下降(P<0.05);热应激下血清中SOD有上升的趋势,但差异不显著(P>0.05);热应激下,血清中MDA浓度升高,应激10～18 h上升显著(P<0.05);应激后2、26 h HSP70基因的相对表达量与应激前相比显著性地上升了5.93倍、2倍(P<0.05),应激后6～22 h HSP70基因的相对表达量与应激前水平无显著性差异(P>0.05)。结果表明,热应激影响锦鲤非特性免疫指标,降低锦鲤非特异性免疫力;HSP70的表达受热应激调节,热应激下诱导合成的HSP70对锦鲤起到一定的应激保护作用。     

Dietary microbial levan ameliorates stress and augments immunity in Cyprinus carpio fry (Linnaeus, 1758) exposed to sublethal toxicity of fipronil

A 45‐day feeding trial was conducted to study the stress ameliorating and immunomodulatory role of microbial levan in Cyprinus carpio fry exposed to sublethal dose (1/10th LC₅₀) of fipronil [(±)‐5‐amino‐1‐(2,6‐dichloro‐α,α,α‐trifluoro‐p‐tolyl)‐4‐trifluoromethylsulfinylpyrazole‐3‐carbonitrile]. Two hundred and twenty‐five fry were randomly distributed in five treatments in triplicates. Four purified diets were prepared with graded levels of microbial levan. Five different treatment groups were levan control L₀P₀ (basalfeed + 0% levan without exposure to pesticide); pesticide control L₀P₁ (basalfeed + 0% levan with exposure to pesticide); L_0.25P₁ (basalfeed + 0.25% levan with exposure to pesticide); L_0.50P₁ (basalfeed + 0.50% levan with exposure to pesticide) and L_0.75P₁ (basalfeed + 0.75% levan with exposure to pesticide). Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and fructose‐1,6‐diphosphatase (FDPase) activites were significantly (P < 0.05) increased, whereas alkaline phosphatase (ALP), adenosine triphosphatase (ATPase) and acetyl choline esterase (AchE) activities were significantly (P < 0.05) reduced in higher levan‐fed groups. RBC, haemoglobin and WBC counts were significantly (P < 0.05) increased in the levan‐fed groups. Similar trends were also observed for the total serum protein, globulin, NBT and lysozyme activities. Blood glucose and serum cortisol exhibited a third order polynomial relationship with increasing level of dietary levan. Overall result showed stress ameliorating, immunostimulating and protective role of microbial levan against fipronil‐induced stress in C. carpio fry at 0.75% level of dietary levan supplementation.     

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

A novel 79‐bp insertion/deletion polymorphism in 3′‐flanking region of IGF‐I gene is associated with growth‐related traits in common carp (Cyprinus carpio L.)

Insulin‐like growth factor‐I (IGF‐I) participates in the modulation of the cellular development, differentiation and proliferation in various vertebrate tissues. Therefore, variations in IGF‐I gene are good candidates as growth regulators. In this study, a novel 79‐bp insertion/deletion (I/D) polymorphism was identified in 3′‐flanking region of IGF‐I gene by polymerase chain reaction (PCR) and sequencing approaches in common carp Cyprinus carpio. To gain insight into the association of the 79‐bp I/D polymorphism with growth‐related traits, a total of 747 individuals from Heilongjiang carp Cyprinus cario haematopterus (YL) (n = 263), selective line of German mirror carp Cyprinus carpio L. mirror (JL) (n = 229), and cold‐resistance line of Hebao red carp Cyprinus carpio var. wuyuanensis (HL) (n = 255) were genotyped. The DD genotype of the polymorphism was significantly associated with higher body length (135), body weight (325, 385, and 445 days), net weight (445 days) and lower overwintering body loss rate in JL. In addition, in pooled population (YL+JL+HL), fish with DD genotype also showed significantly higher body weight (445 days) and net weight (445 days) than those with II genotype. Taken together, these results suggest that the novel polymorphism in IGF‐I gene could be considered as a potential genetic marker for selection of growth performance in common carp.     

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.     

Effect of dietary soybean lecithin on growth parameters,digestive enzyme activity,antioxidative status and mucosal immune responses of common carp (Cyprinus carpio)

Common carp, Cyprinus carpio (13.54 ± 0.48 g), were fed soybean lecithin‐incorporated diets for 8 weeks. After the feeding trials, the different groups of fishes were assessed by survival, growth parameters, digestive enzymes, antioxidant enzyme activities, mucosal immune parameters, etc. The lecithin‐incorporated diets (10–30 g kg^–1) helped to increase the weight gain significantly (p < .05) compared with control groups; activity of digestive enzymes was also significantly (p < .05) higher compared with experimental groups. The antioxidant enzymes including catalase and serum superoxide dismutase were significantly (p < .05) higher in the C. carpio fed lecithin‐incorporated feed than the ones fed the control feed. The serum thiobarbituric acid‐reactive substances and serum glutathione peroxidase activity did not differ significantly (p > .05) between control and experimental groups. The positive correlation supported that antibacterial activity was significantly (p < .05) increased with increasing concentration of lecithin in the diets. Among the different mucosal immune parameters, lysozyme, protease and esterase activities were significantly (p < .05) increased with lecithin‐incorporated diets. The results of this study confirm that 20 and 30 g kg^–1 lecithin inclusion in diets helped to enhance the activities of digestive enzymes, antioxidant enzymes, antibacterial factors and mucosal immune parameters in C. carpio.     

Modulation of growth performances,survival, digestive enzyme activities and intestinal microbiota in common carp (Cyprinus carpio) larvae using short chain fructooligosaccharide

This study investigates the effects of inclusion of low levels of dietary short chain fructooligosacchairde (sc‐FOS) on physiological response and intestinal microbiota of carp (Cyprinus carpio) larvae. After acclimation, fish (550 ± 20 mg) were allocated into nine tanks (40 fish per tank) and triplicate groups were fed a control diet (0%) or diets containing 0.5% and 1% sc‐FOS for 7 weeks. At the end of the experiment, the growth performance parameters (final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and condition factor (CF), survival rate as well as digestive enzyme activities (amylase, lipase and protease), total viable counts of heterotrophic aerobic bacteria (TVC) and lactic acid bacteria (LAB) level in intestinal microbiota were measured. Our results revealed no significant (P > 0.05) effects of sc‐FOS on growth performance and TVC when compared with the control group. However, administration of low levels of dietary sc‐FOS significantly increased digestive enzyme activities (lipase and amylase) and LAB levels (P < 0.05). Also, survival rate was significantly elevated in sc‐FOS fed carp. These results revealed that administration of low levels of sc‐FOS can be considered as a beneficial dietary supplement for larval stage of common carp.     

Sesbania aculeata leaf meal as replacer of de‐oiled rice bran in aquaculture feed: Growth,IGF‐1 expression,metabolic and biochemical responses in Cyprinus carpio (Linnaeus 1758)

A feeding trial was conducted to evaluate the effect of inclusion of Sesbania leaf meal in the diet of Cyprinus carpio as a replacer of de‐oiled rice bran (DORB). Bacillus subtilis was used to ferment the Sesbania leaf meal (SLM) to produce FSLM (fermented sesbania leaf meal). Fishes were fed with five isonitrogenous (30% CP) and isocaloric diets for 60 days and treatments were viz., C (30% DORB, 0% SLM), R15 (15% DORB, 15% SLM), R30 (0% DORB, 30% SLM), F15 (15% DORB, 15%FSLM) and F30 (0% DORB, 30% FSLM) by replacing DORB with SLM. A similar growth performance with control was observed in 15% FSLM group while there was a growth reduction in all the raw leaf meal fed groups. Similarly, the expression of insulin‐like growth factor‐1 (IGF‐1) was significantly (p < .05) higher in control and 15% FSLM fed groups. Growth and metabolic enzymes such as protease, amylase, ALT and AST activities were significantly higher in the fermented leaf meal fed groups compared to their non‐fermented counterparts. Antioxidant enzymes, serum transaminase enzymes (SGOT, SGPT) and total bilirubin (TBR) were found to be increased by enhanced level of RSLM, however, reduced by inclusion of FSLM. However, 30% inclusion level of FSLM even after fermentation showed detrimental effect on growth and metabolic response at cellular and molecular level. Thus, the results concluded that fermented Sesbania leaf meal (FSLM) using B. subtilis could replace 50% DORB (15% FSLM group) in the diet of C. carpio without affecting the growth and other physiological responses.     

Effect of acclimation temperature on thermoregulatory behaviour,thermal tolerance and respiratory metabolism of Lutjanus guttatus and the response of heat shock protein 70 (Hsp70) and lactate dehydrogenase (Ldh‐a) genes

This study evaluated the effect of different environmental temperatures in the physiology of Lutjanus guttatus juveniles by analysing their thermoregulatory behaviour, thermal tolerance, oxygen consumption rates and thermal metabolic scope. Jointly, the effect of acclimation and critical temperatures on heat shock protein 70 (Hsp70) and lactate dehydrogenase (Ldh‐a) gene expressions were also analysed using acclimation temperatures of 20, 23, 26, 29 and 32°C. The results showed that the final preferred temperature in juvenile snapper was 26°C with a thermal window of 336.5°C², which was related to an optimal temperature for their physiology determined by the routine metabolic rate and thermal metabolic scope. At temperatures from 20 to 26°C, the routine metabolic rate and Hsp70 and Ldh‐a genes had the lowest values related to a basal expression level. At acclimation temperatures from 29 to 32°C and after critical thermal maximum (CT_max) limit, the relative expression of Hsp70 and Ldh‐a genes increased significantly, but the main response at CT_max was the upregulation of Hsp70 gene.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.     

Effects of α‐ketoglutarate on growth performance,antioxidant capacity and ammonia metabolization against chronic carbonate alkalinity stress in Songpu mirror carp (Cyprinus carpio Songpu)

A 3 × 2 factorial experiment was conducted to investigate the interaction between carbonate alkalinity and dietary α‐ketoglutarate (AKG) levels on the growth performance, antioxidant capacity and ammonia metabolization of Songpu mirror carp (Cyprinus carpio Songpu). Each diet (0%, 1% AKG) was randomly allotted to 0 mmol/L, 15 mmol/L, 30 mmol/L carbonate alkalinity groups with three replicate aquaria. The weight gain rate (WGR) significantly increased in the 1% AKG group and significantly decreased with increasing carbonate alkalinity (p < .05). Crude ash was significantly affected by the interaction between carbonate alkalinity and dietary AKG levels and significantly increased in the 1% AKG group (p < .05). A significant interaction between carbonate alkalinity and dietary AKG levels on superoxide dismutase (SOD) activities in hepatopancreas was observed (p < .05). The increasing carbonate alkalinities significantly decreased SOD activities in the hepatopancreas and foregut, catalase (CAT) activities in the hepatopancreas, foregut, midgut and hindgut and glutathione (GSH) contents in the hepatopancreas and foregut (p < .05). Despite AKG addition, the blood ammonia content still increased with increasing carbonate alkalinities. The gene expressions of AQP3, Rhag, Rhcg2 and Na⁺/K⁺‐ATPase (NKA) in the gills and glutamine synthase (GS) in the brain were significantly upregulated with increasing carbonate alkalinities (p < .05). In the 1% AKG group, GS gene expression significantly upregulated in the brains, whereas AQP3, Rhag, Rhcg2 and NKA gene expressions significantly downregulated in the gills (p < .05). In summary, the 1% AKG addition can enhance the gene expression of ammonia metabolization and improve the antioxidant capacity of Songpu mirror carp with chronic carbonate alkalinity stress.     

Alteration of the haemocyte parameters,expression of immune‐related and neurohormone bursicon genes of giant river prawn Macrobrachium rosenbergii (de Man) in the response to salinity stress and ammonia stress

We examined the effects of salinity stress and ammonia stress on alteration of the haemocyte count, phenoloxidase activity, expressions of immune‐related genes including prophenoloxidase (proPO), crustin, heat shock protein 70 (HSP70), and expressions of stress‐responsive neurohormone (Bur‐α and Bur‐β) in the thoracic and abdominal ganglia of giant river prawn Macrobrachium. These parameters of prawn that subjected to salinity stress (transferred from 0‰ to 5‰ and 10‰), and subjected to ammonia‐nitrogen (ammonia‐N) stress (transferred from 0 to 0.262 mg/L and 0.786 mg/L) were examined after 0, 3, 6, 24, 72 and 168 hr respectively. During the initial period of 3, 6 and 24 hr, granulocyte haemocyte (granular and semi‐granular hemocyte) count and PO activity significantly decreased, while expressions of Bur‐α and Bur‐β significantly increased. After 24 hr, granulocyte haemocyte count and PO activity significantly increased, whereas expressions of Bur‐α and Bur‐β significantly decreased. The expressions of proPO, crustin and HSP70 were significantly downregulated in the prawn that subjected to salinity stress and ammonia‐N stress at all time periods of 3–168 hr. In conclusion, changes in the granulocyte haemocyte count of M. rosenbergii following salinity stress and ammonia‐N stress are closely associated with the changes of Bur‐α and Bur‐β expressions.     

Effects of l‐tryptophan on the performance,energy partitioning and endocrine response of Japanese sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress

The effects of dietary tryptophan on growth performance, energy budget and endocrine response of sea cucumber Apostichopus japonicus (Selenka) were studied, to test whether the tryptophan could mitigate the crowding stress response of the sea cucumber. Four density treatments of the sea cucumber (i.e. 4, 8, 16 and 32 individuals per 40 L water, represented as L, ML, MH and H) were fed with diets containing 0 (control), 1%, 3% and 5% l ‐tryptophan, respectively, for 75 days. The results showed that the specific growth rate (SGR) and feed conversion ratio (FCE) of A. japonicus decreased with increasing in the stocking densities but increased after supplementation of 1% and 3% tryptophan. The energy allocation of the A. japonicus was affected significantly by stocking density and dietary tryptophan treatments (p < .05). In general, the energy consumed (C) and energy used for growth (G) decreased with the increasing in the stocking densities. The highest proportion of energy deposited to growth occurred in each 3% treatment (L, 6.38%; ML, 6.12%; MH, 4.56% and H, 4.14%), which were significantly higher than the controls (p < .05) respectively. Higher cortisol, glucose and lactate levels of A. japonicus were recorded in the control treatments of MH and H, but l ‐tryptophan supplementation could reduce the levels of cortisol, glucose and lactate of the test animals. The present results proved that the growth performance of the sea cucumber was inhibited by high stocking densities, while dietary supplementation of tryptophan could significantly mitigate the crowding stress, and improve the growth of sea cucumber.