Immune responses of Litopenaeus vannamei to non‐ionic ammonia stress: a comparative study on shrimps in freshwater and seawater conditions

To investigate the effect of non‐ionic ammonia (NH₃‐N) stress (0.1 and 0.5 mg L^?1) on the immunity of Litopenaeus vannamei cultured in long‐term freshwater, the total haemocyte count (THC), the activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase dismutase (SOD) and the content of malondialdehyde (MDA) were determined and further compared with those of seawater shrimps. The results showed that NH₃‐N stress significantly reduced THC and the activity of PO and SOD (P < 0.05). Under 0.1 mg L^?1 NH₃‐N stress, NOS activity increased first and then decreased significantly, while it dropped dramatically under 0.5 mg L^?1 NH₃‐N stress (P < 0.05). During NH₃‐N stress, MDA content increased continuously, and the MDA content in hepatopancreas of freshwater shrimps was higher than that of seawater shrimps. It was concluded that NH₃‐N stress significantly influenced the non‐specific immunity and could also upset the balance of antioxidant system of L. vannamei in both freshwater and seawater shrimps. Compared with in seawater, the shrimps in freshwater were more vulnerable to NH₃‐N stress because of higher lipid peroxidation and lower immunity.     

Alleviation effect of dietary cerium and its complex with chitosan oligosaccharide on cadmium accumulation in juvenile turbot,Scophthalmus maximus L., under cadmium stress

An 8 weeks feeding trial was conducted to investigate the effect of dietary cerium (Ce) and its complex with chitosan oligosaccharide (COS‐Ce) on growth performance and cadmium (Cd) accumulation of turbot, Scophthalmus maximus L. under Cd stress. The basal diet (Diet 0) was formulated without Cd and cerium as the control. Seven other experimental diets (Diets 1–7) were formulated with supplementation of 50 mg Cd²⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 100 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 200 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg COS‐Ce/kg feed, 50 mg Cd²⁺/kg and 100 mg COS‐Ce/kg feed, and 50 mg Cd²⁺/kg +200 mg COS‐Ce/kg feed. Results of the present study showed that, compared with the control group, the condition factor in fish fed the diet with 50 mg Cd²⁺/kg feed (Diet 1) was significantly lower, whereas the Cd concentration in liver and kidney of fish fed the diet with 50 mg/kg Cd²⁺ (Diet 1) was significantly higher (P < 0.05). The high Cd accumulation of fish fed diets with sole 50 mg/kg Cd (Diet 1) could be significantly decreased by 51.72% after supplementation of 200 mg COS‐Ce/kg in the diet (Diet 7). These results suggested that elevated COS‐Ce could effectively protect against dietary Cd accumulation in turbot.     

Effect of dietary β‐1,3‐glucan on the immune response of Litopenaeus vannamei exposed to nitrite‐N

β‐1,3‐Glucan with 1.0 g kg^?1 was supplemented to a basal diet [control (C)] with different feeding schedules: permanently β‐1,3‐glucan diet (BG) and 14 days BG–14 days control diet (C + BG). Growth and immunological responses [respiratory burst (RB), superoxide dismutase (SOD), catalase (CAT), lysozyme and total protein] in haemolymph, hepatopancreas and muscle of Litopenaeus vannamei were recorded after 84‐day feeding and exposure to nitrite‐N (20 mg L^?1) for 120 h. β‐1,3‐Glucan administration did not affect growth performance. However, as compared with control, elevated CAT and lysozyme activities were seen in haemolymph of both BG groups, while significantly higher activities of SOD, lysozyme and RB in hepatopancreas, and higher activities of CAT and lysozyme in muscle were only seen in C + BG group. After nitrite‐N stress, significantly higher haemolymph protein, and hepatopancreas activities of lysozyme and RB were observed in both BG groups, but significantly higher activities of haemolymph SOD was only seen in C + BG group. The mortality in groups BG and C + BG was significantly lower than that in group C, but C + BG group showed a trend of higher nitrite‐N resistance compared with BG group. Considering dietary cost and immunostimulatory effects, the feeding schedule with 14 days BG–14 days control diet is more recommended.     

Protective effects of dietary selenium on abalone Haliotis discus hannai against the toxicity of waterborne cadmium

This study was conducted to investigate protective effects of dietary selenium (Se) on abalone Haliotis discus hannai Ino against the toxicity of cadmium (Cd). A 60‐day feeding trial was conducted with abalone (initial weight: 3.17 ± 0.01 g), which were exposed to 0.34 mg/L of waterborne Cd. During a feeding trial, abalone were fed graded levels of Se at 0.10 (controls), 1.31, and 4.20 mg/kg diet respectively. Results showed that there was no significant difference in specific growth rate and survival rate of abalone among the three treatments. Compared with the controls, dietary Se significantly decreased Cd concentrations in serum, muscle, mantle, gill, mantle, and hepatopancreas of abalone. Besides, compared with the controls, dietary Se significantly increased metallothionein concentration in the hepatopancreas of abalone. Additionally, compared with the controls, dietary Se significantly decreased concentrations of malondiadehyde and protein carbonyl in hepatopancreas of abalone. Meanwhile, compared with the controls, dietary Se significantly increased activities of glutathione peroxidase, thioredoxin reductase, and thioredoxin peroxidase, and concentration of glutathione in the hepatopancreas of abalone. Based on the data above, in abalone, dietary Se showed protective effects against waterborne Cd.     

Dose‐dependent protective effects of dietary selenium on abalone Haliotis discus hannai Ino against the toxicity of waterborne copper

This study was designed to assess the protective effects of dietary selenium (Se) on abalone Haliotis discus hannai Ino against the toxicity of waterborne copper (Cu). A 60‐day feeding trial was conducted in a static water system for abalone (initial weight: 3.17 ± 0.01 g) exposed to 0.02 mg L^?1 of waterborne Cu. The animals were fed one of the three experimental diets with 0.10, 1.31 and 4.20 mg kg^?1 of Se from Na₂SeO₃·5H₂O respectively. Results showed that the abalone fed 1.31 mg kg^?1 of dietary Se had the lowest Cu concentration in shell, muscle, mantle, gill, hepatopancreas and serum. Meanwhile, the significant lowest contents of malondiadehyde and protein carbonyl in hepatopancreas were also found in the treatment with 1.31 mg kg^?1 of dietary Se (P < 0.05). In addition, this treatment had significant higher glutathione content and thioredoxin reductase activity in hepatopancreas (P < 0.05). However, the activity of Se‐dependent glutathione peroxidase (Se‐GPx) was significantly decreased in the treatment with 4.20 mg kg^?1 of dietary Se (P < 0.05). In this treatment, the protein carbonyl content in hepatopancreas was significantly higher than that in the group with 1.31 mg kg^?1 of dietary Se (P < 0.05). In conclusion, in terms of anti‐oxidation and Cu accumulation, the protective effects of dietary Se on abalone against waterborne Cu were dose‐dependent. The 1.31 mg kg^?1 of dietary Se had this effect, but not 4.20 mg kg^?1 of dietary Se. Moreover, the latter increased the oxidative stress in abalone exposed to the waterborne Cu.     

饲料中Pb和Cd在中华绒螯蟹体内的吸收与释放特性

为获知饲料中重金属与中华绒螫蟹各组织间的富集与释放特性,应用生物富集双箱动力学模型,模拟中华绒螫蟹分别在Pb含量为10.21、22.01、40.81 mg/kg,Cd含量为1.78、2.80、4.48 mg/kg的饲料驯养过程中,其鳃、肝胰腺和肌肉对Pb和Cd的生物富集与释放特性,为Pb与Cd在中华绒螫蟹体内的分布、富集和迁移提供理论依据,为中华绒螫蟹安全生产提供指导。同时通过非线性拟合得到中华绒螫蟹对饲料中Pb和Cd的富集速率常数k_1、排出速率常数k_2、生物富集系数BCF、生物半衰期B_(1/2),富集平衡时生物体内Pb和Cd含量CAmax等动力学参数。结果显示:①中华绒螫蟹对饲料中的Pb和Cd具有明显的富集,蟹鳃、肝胰腺和肌肉中Pb的含量与富集时间和饲料中Pb的添加量表现出了很好的正相关,在富集的第48天,各组织器官中Pb的含量达到最大,在鳃中的含量分别为0.18、1.14、1.27和1.91 mg/kg;肝胰腺中含量分别为1.00、2.17、2.33和3.50 mg/kg;肌肉中含量分别为0.18、0.73、1.00和1.35 mg/kg。鳃和肝胰腺对饲料中Cd的吸收与Pb情况类似,在富集的第48天,4个饲料组蟹鳃中的浓度值均达到最高,分别为0.026、0.073、0.107和0.154mg/kg;肝胰腺除了在C组实验的第24天含量达到最高,为1.90mg/kg外,其他3组实验,在富集的第48天含量达到最高分别为0.33、1.05和1.24 mg/kg,C组在第48天的含量有所降低,为1.76 mg/kg。但是肌肉中Cd含量没有明显的规律。②中华绒螫蟹对Pb和Cd的生物富集和释放都较缓慢。达到平衡状态时,鳃、肝胰腺、肌肉各组织器官中Pb含量分别为1.07~1.69、4.87~4.95、0.79~1.28 mg/kg,鳃、肝胰腺中Cd含量分别为0.06~0.14和1.25~2.66 mg/kg。Pb和Cd在组织器官中的生物富集系数(BCF)范围分别为0.03~0.48和0.03~0.87,中华绒螫蟹对Cd的富集能力明显高于Pb;Pb和Cd在各组织器官的生物学半衰期(B_(1/2))范围分别为9~67 d和8~48 d。中华绒螫蟹对Pb的排除能力明显低于Cd。③Pb和Cd在中华绒螫蟹组织器官中的富集具有选择性,在经不同含量Pb和Cd的饲料驯养后得到统一含量分布规律:肝胰腺鳃肌肉。     

iTRAQ‐based identification of differentially expressed proteins related to growth in the swimming crab,Portunus trituberculatus

The swimming crab, Portunus trituberculatus, is an important farmed species in China, and it has been studied extensively, which requires more information on its genetic background. To date, information on the proteomics of P. trituberculatus is scarce. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomics to investigate growth regulation in P. trituberculatus. Total proteins were isolated from five tissues (eyestalk, gill, heart, hepatopancreas and muscle). Equal quantities of protein from each tissue were pooled at the proteome level using the iTRAQ method. A total of 961 proteins were identified using liquid chromatography coupled with tandem mass spectrometry and de novo sequencing data. Using a 1.2‐fold change in expression as a physiologically significant benchmark, 30 differentially expressed proteins (DEPs) were found to undergo differential expression related to the growth of P. trituberculatus, and most of the growth‐related proteins, including those involved in metabolism, immune responses, DNA duplication and protein synthesis, were upregulated, indicating that conservation of energy is an important strategy to cope with growth. There was a high consistency between the expression levels determined using iTRAQ and mRNA, highlighting the high reproducibility of our proteomic approach and its great value in elucidating the molecular mechanisms of P. trituberculatus.     

Effect of oxidized fish oil and α‐tocopherol on growth,antioxidation status,serum immune enzyme activity and resistance to Aeromonas hydrophila challenge of Chinese mitten crab Eriocheir sinensis

The impact of dietary α‐tocopherol on juvenile Chinese mitten crab Eriocheir sinensis was experimentally evaluated in a 10‐week study. Crab were fed with nine diets including three levels of α‐tocopherol (0, 100 and 300 mg kg^?1 diet) and three levels of fish oil oxidation (fresh, moderate and high) in triplicates. Fresh and moderate oil oxidization enhanced weight gain, but moderate and high oil oxidization lowered survival and feed efficiency. The 100‐mg α‐tocopherol kg^?1 diet resulted in higher hepatopancreas MDA than other α‐tocopherol diets. High oil oxidization led to the lowest serum superoxide dismutase (SOD) and glutathione peroxidase (GPH‐PX). The serum SOD and GPH‐PX activities in crab fed 100 mg α‐tocopherol were higher than in those fed other α‐tocopherol diets. The diet without α‐tocopherol addition lowered lysozyme and phenoloxidase (PO) activities compared to other α‐tocopherol diets. Fresh fish oil diet increased PO activity compared to oxidized oils. High oil oxidization caused significantly more mortality than fresh or moderate oxidization after 7‐d postchallenge with Aeromonas hydrophila. Supplementation with α‐tocopherol significantly enhanced resistance to bacterial infection. This study indicates that α‐tocopherol can protect lipid from peroxidation and enhance disease resistance.     

Changes to the histological gill structure and haemolymph composition of early blue swimmer crab Portunus pelagicus juveniles during elevated ammonia‐N exposure and the post‐exposure recovery

It is yet unclear whether sub‐lethal ammonia‐N levels cause irreparable damage to aquatic crustaceans, or if recovery is possible, the potential factors involved. The aim was to investigate the effect of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure on the haemolymph osmolality, Na⁺, K⁺, Ca²⁺, pH, ammonia‐N, total haemocyte counts (THC) and gill histopathology of Portunus pelagicus juveniles at 0, 3, 6, 12, 24 and 48 h respectively. Following 48 h, crabs were transferred to pristine seawater allowing a recovery period up to 96 h and similarly measured. In addition moribund crabs, induced from lethal ammonia‐N levels of 7.036 and 10.518 mmol L^?1, were measured for haemolymph osmolality/ions and pH levels. The results demonstrate that despite severe gill damage within 6‐ and 1 h of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure, respectively, no significant change (P>0.05) in the haemolymph osmolality, Na⁺, K⁺, Ca²⁺ or pH levels occurred or by ammonia‐N‐induced morbidity. Although the gills can completely recover within 24 and 48 h post exposure to 0.706 and 2.798 mmol L^?1 ammonia‐N, respectively, likely facilitated by significant haemocyte increases (P<0.05) within the haemolymph and gill lamellae, dependent factors were the previous ammonia‐N concentration and recovery duration while individual variability was also noticed.     

1H‐NMR metabolomic profiling of the crayfish Astacus leptodactylus subjected to polyphenol‐enriched diets

¹H‐NMR analysis of the hepatopancreas, muscle and haemolymph of Astacus leptodactylus after feeding with polyphenol‐enriched diet is reported. ¹H‐NMR spectra of lipophilic extracts showed the presence of cholesterol, fatty acid residues, phospholipids and triglycerides. ¹H‐NMR spectra of aqueous extracts identified 35 metabolites in the hepatopancreas, 31 in the muscle and 22 in the haemolymph. A total of 20 metabolites (amino acids and their derivatives) were present in the hepatopancreas, the muscle and the haemolymph. A total of 10 metabolites were present in both the hepatopancreas and the muscle (five amino acids, 2‐hydroxybutyrate, choline, myo‐inositol, glycogen and uracil). 2‐Hydroxyisobutyrate and creatine were present in both the hepatopancreas and the haemolymph. Phosphorylethanolamine, phosphocholine and fumarate were present only in the hepatopancreas and isoleucine only in the muscle. Statistical analysis showed that the percentage of weight gain was statistically higher in polyphenol‐enriched diet groups compared to the control and that polyphenols had a stimulating effect on the general metabolism. No stress‐related metabolites were higher in crayfish fed with polyphenol‐enriched diet. Conversely, phosphatidylcholine, cholesterol and DHA, linked to resistance to environmental stress and diseases, were higher compared to the control diet. This study indicates that ¹H‐NMR is a useful tool to study the metabolomics in relation to diet differences.     

Mechanism of Cd<Superscript>2+</Superscript> on DNA cleavage and Ca<Superscript>2+</Superscript> on DNA repair in liver of silver crucian carp

The subject of acute injury, apoptosis and canceration of animals induced by heavy metal ions has been one of the hotspots studied worldwide. However, the exact molecular mechanism of Cd-induced carcinogenicity remains largely unclear, and how to relieve the toxicity in vivo has rarely been reported. For this paper, we have investigated the mechanism of Cd²⁺ on DNA cleavage and Ca²⁺ on DNA repair in the liver of silver crucian carp (Carassius auratus gibelio) by agarose gel electrophoresis methods and by estimating biochemical indexes. Our results show that Cd²⁺ induces the classical laddering degradation of DNA in vivo and that DNA cleavage is repaired after injection with Ca²⁺ under various Cd²⁺ concentrations. DNA cleavage caused by Cd²⁺ is due to the activation of deoxyribonuclease (DNase) and the accumulation of reactive oxygen species (ROS). Furthermore, Cd²⁺ destroys the antioxidant system, which diminishes the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), causing an increase of the lipid peroxidation (LPO) level, respectively. However, after the liver is injected with Ca²⁺ under various Cd²⁺ concentrations, the DNase activity, the ROS generating rate and the LPO level are obviously reduced, the activities of SOD, CAT, and POD are greatly increased. At the same time, ROS production and removal recoves its balance. The results show that Ca²⁺ can relieve the toxicity of Cd²⁺ in silver crucian carp.     

Effect of dietary l‐carnitine on growth performance and antioxidant response in Amur minnow (Phoxinus lagowskii Dybowskii)

l ‐carnitine (LC) is required for transporting long‐chain fatty acids into the mitochondria, where β‐oxidation takes place, and it works as an antioxidant molecule against reactive oxygen species. This study evaluated the effects of LC on the growth and antioxidant function of Amur minnow (Phoxinus lagowskii Dybowskii). Five isonitrogenous (380.4 g/kg) and isoenergetic (17.63 MJ/kg) diets were supplemented with five LC levels: control level (0 mg/kg) and treatment levels (50, 400, 750, or 1,100 mg/kg) were fed to fish (18.19 ± 0.56 g) for 120 days. The results showed that the growth performance of fish fed a diet containing 400 mg/kg of LC was significantly higher than that of the control and those fed other LC level treatments. Similarly, the 400 mg/kg treatment had the best feed efficiency. Further, the levels of total antioxidant capacity and total glutathione in the serum and hepatopancreas of fish fed a diet containing 750 mg/kg of LC were significantly increased; however, malondialdehyde levels were significantly reduced compared to those of the control group. The activities of antioxidant enzymes of 750 mg/kg treatments in the serum and hepatopancreas were significantly higher than those of the control group, including total superoxide dismutase, catalase, glutathione peroxidase and gamma‐glutamyl‐cysteine synthetase. Finally, 750 mg/kg treatment significantly upregulated the mRNA relative expression of antioxidant enzymes and nuclear factor erythroid‐2‐related factor 2 and inhibited the mRNA level of kelch‐like ECH‐associated protein 1 in the hepatopancreas. In conclusion, the dietary LC level of 400–750 mg/kg could improve the growth performance, feed utilization and antioxidant defense system of Amur minnow under the culture conditions.     

Tissue distribution of hepatopancreatic parvo‐like virus of shrimp in freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of hepatopancreatic parvo‐like virus (HPV) of shrimp in different organs of freshwater rice‐field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT‐PCR, ELISA, Western blot and q‐PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large‐scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT‐PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q‐PCR. The results revealed a steady decrease in C_T values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post‐larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice‐field crab could be used as an alternative host for HPV replication and also for large‐scale production of HPV.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Dietary nano‐selenium enhances antioxidant capacity and hypoxia tolerance of grass carp Ctenopharyngodon idella fed with high‐fat diet

To evaluate the effects of dietary nano‐selenium (Nano‐Se) on antioxidant capacity and hypoxia tolerance of grass carp fed with high‐fat diet, experimental fishes were fed Nano‐Se supplemented diets at doses of 0 (Control), 0.3, 0.6, 0.9 and 1.2 mg/kg for 10 weeks. After feeding trial, a part of the fishes were exposed to hypoxia stress. Results showed that the survival ratio of grass carp significantly increased in 0.6 and 0.9 mg/kg Nano‐Se group, and the content of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) significantly decreased in 0.6–1.2 mg/kg Nano‐Se groups compared with the control group. In addition, dietary Nano‐Se significantly enhanced glutathione peroxidase (GPX) activity and reduced the malondialdehyde (MDA) content in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. Dietary Nano‐Se significantly elevated mRNA expression of GPX1 and catalase (CAT) by promoting the mRNA expression of NF‐E2‐related nuclear factor 2 (Nrf2) in the hepatopancreas. After hypoxia stress, the GPX and superoxide dismutase (SOD) activities were significantly enhanced, and the MDA content and mortality rate consequently decreased in fishes fed diets with 0.3 and 0.6 mg/kg Nano‐Se. In summary, these results suggested that optimal Nano‐Se in diet enhanced the antioxidant capacity and hypoxia tolerance of grass carp.