Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 μg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 μg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.     

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.     

Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass,Lates calcarifer challenged with Vibrio harveyi

The present study was aimed to address the possible evaluation of Azadirachta indica (neem) leaf-supplemented diets on innate immune response in Asian seabass, Lates calcarifer fingerlings against Vibrio harveyi infection. Fish were fed for two weeks diets containing six graded levels of neem leaf at 0 g, 1 g, 2 g, 3 g, 4 g and 5 g per kg feed. Fish fed neem leaf-supplemented diet displayed significant differences (p < 0.05) in weight gain, specific growth rate (SGR) and feed conversion ratio (FCR) compared to the control group fed without neem leaf-supplemented diet. Various innate immune parameters were examined pre-challenge and post-challenge. Fish was injected intraperitoneally with a lethal dose of V. harveyi containing 10⁸ cells mL^?1. Supplementation of neem leaf diet significantly increased phagocytic activity, superoxide anion production, serum lysozyme, serum bactericidal activity, serum anti-protease activity throughout the experimental period when compared with the control group. Dietary doses of neem leaf diet significantly influenced the immune parameters, haematological parameters and blood biochemical indices of treated fish. The results suggested that fish fed neem leaf-supplemented diet improved the immune system and increased survival rate in L. calcarifer fingerlings against V. harveyi infection.     

Dietary administration of zingerone to enhance growth, non-specific immune response, and resistance to Vibrio alginolyticus in Pacific white shrimp (Litopenaeus vannamei) juveniles

Zingerone, one of the active components of ginger, is a phenolic alkanone with antioxidant and anti-inflammatory properties. The effects of zingerone supplementation on the growth, immunity, and disease resistance of Pacific white shrimp (Litopenaeus vannamei) juveniles were studied. Four experimental diets, including a control diet (without zingerone enrichment) and 1, 2.5, and 5 mg zingerone (kg diet)⁻¹ were used. After 56 days of culture, shrimp fed diets supplemented with 1, 2.5, and 5 mg zingerone (kg diet)⁻¹ had significantly greater weight gain and feed efficiency than the controls. Furthermore, after 56 days of culture, shrimp fed all doses of the zingerone diet had higher survival rates compared to the controls after 24-72 h of challenge by the pathogen, Vibrio alginolyticus. Significantly increased phenoloxidase levels were found in shrimp fed the zingerone diets at all doses, and respiratory bursts, lysozyme and phagocytic activities of shrimp fed 2.5 and 5 mg zingerone (kg diet)⁻¹ also significantly increased. Neither the total hemocyte count nor superoxide dismutase activity of the experimental and control groups revealed significant differences at any dose. The results indicate that zingerone can be recommended as a supplement to shrimp feed to increase growth, immunity, and disease resistance against the pathogen, V. alginolyticus. Use of zingerone as appetizer and immunostimulant in shrimp is promising.     

Influence of probiotic bacterium Bacillus cereus isolated from the gut of wild shrimp Penaeus monodon in turn as a potent growth promoter and immune enhancer in P. monodon

A probiotic bacterium isolated from the gut of wild shrimp Penaeus monodon rendered maximum antagonistic activity against shrimp pathogens and was capable of producing extracellular enzymes. The probiotic bacterium was identified as Bacillus cereus through 16S rRNA sequencing. The lyophilized B. cereus was supplemented with shrimp basal diet at four different concentrations (0.1–0.4%/100 g feed) in D1–D4 diets. The viability of probiotic bacterium in the test diets was evaluated during the study period at various time intervals. The viability ranged from 50.24 ± 1.42 to 180.34 ± 1.30 CFU/g in D1 to D3 diets on the 30th day, whereas it was slightly declined from 45.23 ± 1.30 to 169.13 ± 1.18 CFU/g during the 90th day of storage. A control diet (C), devoid of probiotic supplementation was also simultaneously prepared. During experimentation, P. monodon postlarvae (PL-15) were cultured in individual one tonne capacity FRP tanks in triplicates provided with equal amount of substratum (clay soil) and fed with these respective diets at ad libitum for 90 days. Survival was high (82.0 ± 1.60%) in D4 diet fed shrimp as against a low survival of 65.0 ± 1.33% displayed by control diet fed shrimp. Overall growth responses inferred that a maximum production of 10.45 ± 0.275 g, SGR of 4.40 ± 0.179% and a better FCR of 1.27 ± 0.081 were obtained in D4 diet fed shrimp. However, the water quality parameters showed nonsignificant (P > 0.05) variations among the control and the probiotic treated groups. The tested immunological parameters such as Total haemocyte count, phenoloxidase activity, respiratory burst activity, lysozyme activity, plasma protein concentration and bactericidal activity were higher in D4 diet fed P. monodon, when compared to that of other diets fed shrimp. It is therefore suggested that lyophilized probiotic B. cereus at a concentration of 0.4%/100 g feed was efficient in stimulating the growth and immunity in shrimp.     

Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants

To improve the immune response in tiger shrimp Penaeus monodon against WSSV infection, juveniles (350 ± 10 mg) were vaccinated with formalin-inactivated WSSV and fed with herbal immunostimulants. The methanolic extracts of herbal immunostimulants such as Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa, Withania somnifera and Zingiber officinalis were incorporated in formulated diets at different concentrations; 250 (ED(1)), 500 (ED(2)), 1000 (ED(3)) and 2000 (ED(4)) mg kg(-1) of feed and fed for 60 days after vaccination. After 30 and 60 days intervals of feeding, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps fed with control diets (C(1)) succumbed to death within 5 days after WSSV challenge, when no vaccination and immunostimulations were given. The other control groups (C(2) and C(3)) had slight improvements in all parameters including survival. The percentage survival was significantly (P < 0.05) increased to 30, 50 and 60% in the ED(2), ED(3) and ED(4) diets respectively after 60 days challenging. The better haematological, biochemical and immunological parameters were also found in the herbal extracts supplemented diets fed vaccinated shrimps. The present study revealed that the combined effect of immunostimulation and vaccination helped to boost the immune system against WSSV infection and hence this application can be adopted for shrimp culture.     

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.     

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P < 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2 mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3 mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3 mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.     

The effect of fucoidan from brown seaweed Sargassum wightii on WSSV resistance and immune activity in shrimp Penaeus monodon (Fab)

The polysaccharide-fucoidan was extracted from brown seaweed Sargassum wightii and characterized through FT-IR and ¹³C & ¹H NMR analysis. The extracted fucoidan was supplemented with pellet diets at three different concentrations (0.1, 0.2 and 0.3%). The fucoidan supplemented diets were fed to Penaeus monodon for 45 days, then challenged with WSSV and the mortality percentage was recorded daily up to 21 days. During the challenge test, the control group showed 100% mortality within 10 days, but in the experimental groups, the mortality percentage (51-72% within 21 days) was decreased considerably (P < 0.05) with respect to the concentrations of fucoidan. The reduction in mortality percentage of experimental groups over control group was ranged from 50.81 to 68.06%. During challenge experiment, the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity and phagocytic activity were measured before injection of WSSV (0 day) and after the injection of WSSV on 10th and 21st days, respectively. All the immunological parameters of experimental groups were significantly (P < 0.05) increased than control group. RT-PCR analysis confirmed the considerable reduction of WSSV DNA copy numbers with respect to the concentration of fucoidan. It was concluded that P. monodon fed with fucoidan of S. wightii supplemented diet had enhanced the innate immunity and increased resistance against WSSV infection.     

Effect of probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge

Antioxidant defences and induced oxidative stress tissue damage of the blue shrimp Litopenaeus stylirostris, under challenge with Vibrio nigripulchritudo, were investigated for a 72-h period. For this purpose, L. stylirostris were first infected by immersion with pathogenic V. nigripulchritudo strain SFn1 and then antioxidant defences: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), Total antioxidant status (TAS), glutathiones and induced tissue damage (MDA and carbonyl proteins) were determined in the digestive gland at 0, 12, 24, 48 and 72 h post-infection (h.p.i.). In the meantime, TAS was also measured in the blood. Infection level of the shrimps during the challenge was followed by determining V. nigripulchritudo prevalence and load in the haemolymph of the shrimps. Changes in all these parameters during the 72-h.p.i. period were recorded for control shrimps and shrimps previously fed for one month with probiotic Pediococcus acidilactici MA18/5M at 10⁷ CFU g^?1 of feed.Our results showed that immersion with V. nigripulchritudo led to maximal infection level in the haemolymph at 24 h.p.i. preceding the mortality peak recorded at 48 h.p.i. Significant decreases in the antioxidant defences were detected from 24 h.p.i. and beyond that time infection leaded to increases in oxidative stress level and tissue damage. Compared to control group, shrimps fed the probiotic diet showed lower infection (20% instead of 45% at 24 h.p.i. in the control group) and mortality (25% instead of 41.7% in the control group) levels. Moreover, infected shrimp fed the probiotic compared to uninfected control shrimps exhibited very similar antioxidant status and oxidative stress level. Compared to the infected control group, shrimps fed the probiotic sustained higher antioxidant defences and lower oxidative stress level.This study shows that bacterial infection leads to oxidative stress in L. stylirostris and highlighted a beneficial effect of P. acidilactici, suggesting both a competitive exclusion effect leading to a reduction of the infection level and/or an enhancement of the antioxidant status of the shrimps.     

Dietary beta-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus

The effectiveness of dietary beta-1,3-glucan (BG), derived from Schizophyllum commune, in modulating the non-specific immunity of the grass prawn Penaeus monodon and its resistance to white spot syndrome virus (WSSV) were investigated. Juvenile P. monodon (6.5+/-0.4 g) were fed for 20 days on a series of test diets containing graded levels of BG (0, 1, 2, 10, 20 g kg(-1)diet) and were then challenged by injection of WSSV. The haemolymph total haemocyte count (THC), phagocytosis (PI), phenoloxidase (PO), superoxide anion (O(2)(-)) and superoxide dismutase (SOD) production were measured at days 0, 1, 3, 6, 9, 12 and 24 after challenge, and shrimp survival rate was also recorded. All the shrimps fed on diets containing BG no more than 1 g kg(-1)died by day 12. Conversely, the survival rate of shrimp fed with the diet containing 10 g kg(-1)BG was significantly higher (P<0.05) by day 9 than that of the other groups. When screened by the WSSV PCR diagnostic procedure, the percentages of surviving juveniles of the BG 2, 10, 20 g kg(-1)groups that were 2-step WSSV negative, were 55, 65 and 65%, respectively. The haemolymph THC, PO, O(2)(-)and SOD production of the 2, 10 and 20 g kg(-1)BG diet groups dropped drastically immediately after the WSSV challenge but subsequently returned to normal. Therefore, oral administration of BG at an optimal level of 10 g kg(-1)diet for 20 days effectively enhanced the immune system and improved the survival of WSSV-infected P. monodon.     

Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus vannamei and its resistance against experimental Vibrio alginolyticus infection

In this study, we examined the effect of fucoidan on the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus infection. Fucoidan induced degranulation, caused changes in the cell morphology, and increased activation of prophenoloxidase (proPO) and the production of superoxide anions in vitro. Shrimp that received fucoidan via immersion at 100, 200, and 400 mg l^?1 after 3 h showed haemocyte proliferation and a higher mitotic index of haematopoietic tissue. In another experiment, the haemocyte count, phenoloxidase (PO) activity, and respiratory bursts (RBs) were examined after the shrimp had been fed diets containing fucoidan at 0 (control), 0.5, 1.0, and 2.0 g kg^?1 for 7–21 days. Results indicated that these parameters directly increased with time. The immune parameters of shrimp fed the 1.0 g kg^?1 diet were significantly higher than those of shrimp fed the 2.0 g kg^?1 diet after 14 and 21 days. Phagocytic activity and the clearance efficiency against V. alginolyticus were significantly higher in shrimp fed the 1.0 g kg^?1 diet compared to those of shrimp fed the 0, 0.5 and 2.0 g kg^?1 diets. In a separate experiment, shrimp that had been fed diets containing fucoidan for 21 days were challenged with V. alginolyticus at 10⁶ colony-forming units shrimp^?1. Survival rates of shrimp fed the 1.0 and 2.0 g kg^?1 diets were significantly higher than those of shrimp fed the 0 and 0.5 g kg^?1 diets for 96–120 h. We concluded that fucoidan provokes innate immunity of shrimp as evidenced by haemocyte degranulation, proPO activation, and the mitotic index of haematopoietic tissue, and that dietary administration of fucoidan at 1.0 g kg^?1 enhanced the immune response of shrimp and their resistance against V. alginolyticus infection.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Bacterial clearance rate and a new differential hemocyte staining method to assess immunostimulant activity in shrimp

New methods were developed to assess immunostimulant efficacy in the black tiger shrimp Penaeus monodon. Test shrimp were fed with 2 or 4 % yeast extract (YE)-coated feed while controls were fed non-coated feed. After 4 wk of feeding, individual shrimp were assessed for total hemocyte counts (THC), the number of granular hemocytes (GH) and rate of bacterial clearance. For hemocyte counts, formalin-fixed hemolymph was stained with 1.2 % Rose Bengal in 50 % ethanol for 20 min at room temperature. Some of this mixture was used for THC with a hemocytometer while some was smeared on a microscope slide and left to dry before counterstaining with hematoxylin for GH counts. By this technique, high quality smears were obtained for accurate differential counts. Bacterial clearance assays were used to assess the sum effect of humoral and cellular defense mechanisms. Vibrio harveyi was injected intramuscularly at 1 x 10(8) cells per shrimp and hemolymph was collected in anticoagulant at 0, 15, 30 and 60 min post-injection for quadruplicate drop counts (20 microl) on TCBS agar. Total hemocyte counts for shrimp fed with 4 % YE were significantly higher (p < 0.05) than those for shrimp fed with non-coated feed. The percentage of granular cells and the rates of bacterial clearance for the YE-fed shrimp were higher than those for shrimp fed the control diet. These 2 methods provide a simple and rapid comparison of shrimp groups for differences in anti-bacterial defense capacity.     

Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes

Immunostimulants are the substances, which enhance the non-specific defence mechanism and provide resistance against the invading pathogenic micro-organism. In order to increase the immunity of shrimps against the WSSV, the methanolic extracts of five different herbal medicinal plants like Cyanodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba were selected and mixed thoroughly in equal proportion. The mixed extract was supplemented with various concentrations viz. 100 (A), 200 (B), 400 (C), and 800 (D) mgkg(-1) through artificial diets individually. The prepared diets (A-D) were fed individually to WSSV free healthy shrimp Penaeus monodon with an average weight of 8.0+/-0.5g for 25 days. Control diet (E), devoid of herbal extract was also fed to shrimps simultaneously. After 25 days of feeding experiment, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps succumbed to death within 7 days when fed on no herbal immunostimulant diet (E). Among the different concentrations of herbal immunostimulant supplemented diets, the shrimps fed on diet D (800mgkg(-1)) significantly (P<0.0001) had more survival (74%) and reduction in the viral load. Also the better performance of haematological, biochemical and immunological parameters was found in the immunostimulant incorporated diets fed shrimps. The present work revealed that the application of herbal immunostimulants will be effective against shrimp viral pathogenesis and they can be recommended for shrimp culture.     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

Marine ash‐products influence growth and feed utilization when Atlantic cod Gadus morhua L. are fed plant‐based diets

Atlantic cod Gadus morhua L. were fed high plant protein diets added to either shrimp‐shell meal or crab‐shell meal. The aims were to investigate if diluting dietary energy would reduce the liver index (HSI) and if marine ash would add value to plant protein‐based diets. Two control diets were used: a high plant protein control diet (PP) with no marine ash addition, and a fishmeal‐based diet (FM) with no marine ash addition. All diets were evaluated in small cod (initial weight 79 ± 15g) and in market‐size cod (initial weight 1579 ± 20 g). Addition of crab‐shell meal up to 20% and shrimp‐shell meal up to 10% did not influence liver size in either small or market‐size cod. An addition of up to 20% crab‐shell meal and 10% shrimp‐shell meal improved growth compared to the PP control diet, and stimulated increased feed intake. However, 10% shrimp‐shell meal and 20% crab‐shell meal diets resulted in a similar intake of energy and protein as the control groups. Increasing shrimp‐shell meal to 20% resulted in reductions in feed intake, fat digestibility and growth, and in altered gut histology. All diets, except the 20% shrimp added diet, resulted in normal ranges of plasma nutrients and blood hematological values, showing good fish health with or without the marine ash addition.     

Effect of dietary copper on the growth performance,non-specific immunity and resistance to Aeromonas hydrophila of juvenile Chinese mitten crab,Eriocheir sinensis

An 8-week feeding trial was conducted to determine the dietary copper (Cu) on growth performance and immune responses of juvenile Chinese mitten crab Eriocheir sinensis. Six semi-purified diets with six copper levels (1.88, 11.85, 20.78, 40.34, 79.56 and 381.2 mg kg^?1 diet) of CuSO₄·5H₂O were fed to E. sinensis (0.45 ± 0.01 g). Each diet was fed to the crab in five replicates. The crab fed diets with 20.78 and 40.34 mg Cu kg^?1 diet had significantly greater weight gain and hemolymph oxyhemocyanin content than those fed diets with 1.88 and 381.2 mg Cu kg^?1 diet. Survival rates of crab were not significantly different between all treatment groups. The activities of copper–zinc superoxide dismutase (Cu–Zn SOD), phenoloxidase (PO), and total hemocyte count (THC) significantly increased when the supplementation of dietary copper reached 20.78–40.34 mg Cu kg^?1 diets. In the bacteria challenge experiment with Aeromonas hydrophila, survival rates significantly increased and reached a plateau when the dietary copper increased from 1.88 to 40.34 mg kg^?1, whereas significantly decreased when the dietary copper increased from 40.34 to 381.2 mg kg^?1. This study indicates that the level of dietary copper is important in regulating growth and immune response in crab.