Dietary seaweed (Enteromorpha) polysaccharides improves growth performance involved in regulation of immune responses, intestinal morphology and microbial community in banana shrimp Fenneropenaeus merguiensis.

The present study was conducted to evaluate the effects of marine polysaccharides from seaweed Enteromorpha on growth performance, immune responses, intestinal morphology and microbial community in the banana shrimp Fenneropenaeus merguiensis. Two thousand and four hundred juvenile shrimps with an average body weight of 2.18 ± 0.06 g were fed for 42 d with diets containing different levels of Enteromorpha polysaccharides (EPS): 0 (control), 1, 2 and 3 g/kg as treatment groups, each of group was replicated three times with two hundred shrimps per replicate. Dietary supplementation of 1 g/kg EPS showed a consistent improvement in the final weight, weight gain, average daily gain rate (ADGR) and specific growth rate (SGR) (P < 0.05), while showed a decrease in the feed conversion ratio (FCR) of shrimp (P < 0.05). Besides, the total anti-oxidative capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), lysozyme (Lyz), alkaline phosphatase (ALP), and phenoloxidase (PO) activities in hemolymph were enhanced by dietary supplementation of 1 g/kg EPS (P < 0.05), while it reduced the hemolymph MDA content (P < 0.05). Shrimp fed 1 g/kg EPS supplemented diets up-regulated FmLyz, FmSOD5 and FmCLAP gene expression level of hepatopancreas and gill (P < 0.05), and also improved the intestinal FmLC2, FmLyz, FmSOD5 and FmCLAP gene expression levels (P < 0.05). In addition, shrimp fed diets containing 1 g/kg EPS increased the villus width (P < 0.05) and resulted in a higher villus surface area (P < 0.05). According to 16S rRNA sequencing results, dietary supplementation of 1 g/kg EPS tended to increase the relative abundance of Firmicutes at phylum level (P = 0.07) and decrease the relative abundance of Vibrio at genus level (P = 0.08). There was a significant positive correlation between the relative abundance of Firmicutes and mRNA expression of intestinal immune-related genes (P < 0.05). These findings revealed that dietary 1 g/kg EPS could improve growth performance, enhance nonspecific immunity and modulate intestinal function of banana shrimp F. merguiensis.

Dietary supplementation of drumstick tree, Moringa oleifera, improves mucosal immune response in skin and gills of seabream, Sparus aurata, and attenuates the effect of hydrogen peroxide exposure.

In aquatic animals, the mucosal barrier is the first line of innate immune defence against external chemicals and pathogens. In this study, the effects of dietary Moringa oleifera leaf (MOL) supplementation on skin and gill mucosal immunity, antioxidants and stress responses were evaluated in seabream (Sparus aurata) fingerlings exposed to hydrogen peroxide (H2O2). A total of 144 specimens (10.11 ± 0.41 g) were divided into four treatments (three replicates per treatment contained 12 specimens each) and fed a non-supplemented control diet or a 1, 2.5 or 5% MOL-supplemented diet. After three weeks of feeding, six specimens from each aquarium were sampled for blood, mucus and tissues. The other six fish in each aquarium were subjected to H2O2 exposure. The results revealed that MOL did not negatively affect either cortisol or glucose levels. MOL supplementation significantly (P < 0.05) improved skin mucosal immunity-related characteristics, including phosphatase, peroxidase and lysozyme activity and IgM levels. Additionally, MOL upregulated the expression of antioxidant genes (sod and cat), an anti-inflammatory gene (tgf-ß), tight junction protein genes (occludin and zo-1), c3, and igm in both the skin and gills. However, H2O2 exposure significantly (P < 0.05) increased both cortisol and glucose levels and disrupted skin mucosal immune function by significantly (P < 0.05) decreasing phosphatase, peroxidase, protease, antiprotease and lysozyme activity and IgM levels. H2O2 exposure severely decreased the mRNA levels of the studied genes. MOL dietary supplementation at the 5% level successfully attenuated the negative effects of H2O2 on the mucosal immune response in both the skin and gills. In conclusion, dietary MOL supplementation at the 5% level is recommended to improve S. aurata mucosal immune function under both normal and stress conditions. Additionally, exposure to H2O2 disrupts the mucosal immunity of fish. This contributes knowledge on the routes involved in mucosal innate immunity and could help to understand the fish resistance against chemicals exposure. Graphical abstract.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Novel C-type lectin from razor clam Sinonovacula constricta agglutinates bacteria and erythrocytes in a Ca2+-dependent manner.

Among its other physiological roles, C-type lectins functioned as pattern recognition receptors (PRR) in innate immunity received much attention. In the present study, a novel C-type lectin was identified and characterized from the invertebrate razor clam Sinonovacula constrict and designated as ScCTL. The complete cDNA sequence of ScCTL was 828 bp in length and coded a secreted polypeptide of 158 amino acids with a typical CRD domain. Multiple sequence alignments combined with phylogenetic analysis both collectively confirmed that ScCTL was a novel member belong to lectin family. Spatial expression distribution analysis revealed that ScCTL was extensively expressed in all of the examined tissues, and the highest expression was detected in the hepatopancreas. After 1 × 107 CFU/mL Vibrio parahaemolyticus challenge by immersion infection, the ScCTL transcript in hepatopancreas and gill were markedly upregulated and arrived the maximum levels at 24 or 12 h after challenge, respectively. Recombinant ScCTL could agglutinate not only all tested bacteria but sheep and mouse erythrocyte in the presence of Ca2+. All of our studies suggested that ScCTL performed important roles in protecting cells from pathogenic infection in S. constrict.

Methionine hydroxy analogue supplementation modulates gill immunological and barrier health status of grass carp (Ctenopharyngodon idella).

This study was conducted to investigate the effects of methionine hydroxy analogue (MHA) on the physical barrier and immune defence in the gill of young grass carp (Ctenopharyngodon idella). A total 630 young grass carp with an average initial weight of 259.70 ±â€¯0.47 g were fed graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine (DLM) group (6.4 g/kg diet) for 8 weeks. After feeding trial, 15 fish from each treatment were challenged with Flavobacterium columnare. Compared to the basal diet, optimal MHA improved cellular structure integrity of gill via repressing death receptor and mitochondria pathways induced apoptosis, which might be related to the down-regulation of c-Jun-N-terminal kinase mRNA levels (P < .05). Simultaneously, optimal MHA supplementation improved cellular structure integrity of gill via elevating glutathione contents, antioxidant enzymes activities and corresponding isoforms mRNA levels to attenuate oxidative damage, which might be to the up-regulation of NF-E2-related factor 2 mRNA levels and down-regulation of Kelch-like ECH-associating protein 1a mRNA levels (P < .05). Besides, optimal MHA improved intercellular structure integrity of immune organs via up-regulating the mRNA levels of intercellular tight junctions-related genes, which might be owing to the down-regulation of myosin light chain kinase (MLCK) mRNA levels (P < .05). Summarily, MHA could improve the physical barrier of fish gill. In addition, optimal MHA supplementation increased lysozyme (LZ) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M contents and up-regulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin and ß-defensin, suggesting that MHA could enhance antimicrobial ability of fish gill. Meanwhile, optimal MHA supplementation enhanced the immune defence of gill via down-regulating pro-inflammatory cytokines mRNA levels and up-regulated anti-inflammatory cytokines mRNA levels, which might be attributed to the down-regulation of nuclear factor κB p65, c-Rel, IκB kinase ß, p38 mitogen activated protein kinase, eIF4E-binding protein1 (4E-BP1) and 4E-BP2 mRNA levels and up-regulation of inhibitor of κBα, ribosomal protein S6 kinase 1 and target of rapamycin mRNA levels (P < .05). In conclusion, the positive effect of MHA on gill health is associated with the improvement of the defence against apoptosis, antioxidant status, tight junctions and immune defence of fish gill. Meanwhile, MHA was superior to DLM on improving the physical barrier of fish gill. For the direction to healthy breeding of young grass carp, the optimal MHA supplementation levels on the premise of 4.01 g/kg methionine basal were estimated by quadratic regression curve, such as 5.49, 6.17 and 6.02 g/kg diet bases on the defence against gill-rot, malondialdehyde content and LZ activity in the gill, respectively.

Towards the modulation of oxidative damage, apoptosis and tight junction protein in response to dietary leucine deficiency: A likely cause of ROS-induced gill structural integrity impairment.

The current study explored the protective effect of leucine on antioxidant status, apoptosis and tight junction damage in the gill of grass carp (Ctenopharyngodon idella Val.). The trial was conducted by feeding grass carp with six graded level of leucine (7.1, 8.9, 11.0, 13.3, 15.2 and 17.1 g kg-1 diet) for 8 weeks. The fish were fed to apparent satiation 4 times per day. The results indicated that compared with the leucine deficiency group, 8.9-11.3 g leucine kg-1 diet supplementations decreased protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the improvement of antioxidant status in the gill by increasing hydroxyl radical capacity and anti-superoxide radicals, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT), glutathione-S-transferase (GST) and glutathione reductase (GR), that referring to the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression. Moreover, leucine deficiency induced DNA fragmentation via the up-regulation of caspase-3, caspase-8 and caspase-9 expressions and down-regulation of target of rapamycin and ribosomal S6 protein kinase 1 expressions. Furthermore, leucine deficiency increased interleukin-1ß (IL-1ß), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) mRNA expression and decreased IL-10 and transforming growth factor ß (TGF-ß), which was partly related to nuclear factor κB (NF-κB) and its inhibitor (IκB). In contrast, the relative mRNA expression of IL-1, IL-8 and TNF-α was down-regulated with 8.9-11.3 g leucine kg-1 diet supplementations. Finally, the relative mRNA expression of tight junction protein, including occludin, zonula occludens-1, claudin b, claudin 3 and claudin 12 was up-regulated with leucine diet supplementations. Our results indicate that leucine protected the fish gill structural integrity partially because of the inhibition of apoptosis, the improvement of antioxidant status, the regulation of tight junction protein and related signalling molecules mRNA expressions in the fish gill.

Protective role of phenylalanine on the ROS-induced oxidative damage, apoptosis and tight junction damage via Nrf2, TOR and NF-κB signalling molecules in the gill of fish.

This study explored the possible preventive effects of dietary phenylalanine (Phe) on antioxidant responses, apoptosis and tight junction protein transcription in the gills of young grass carp (Ctenopharyngodon idella). Fish were fed six different experimental diets containing graded levels of Phe (3.4-16.8 g kg-1) for 8 weeks. The results showed that Phe deficiency induced protein oxidation and lipid peroxidation by decreasing the glutathione content and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) in fish gill (P < 0.05). These results may be ascribed to the downregulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1), and the upregulation of Kelch-like-ECH-associated protein 1 a (Keap1a) expression in grass carp gills (P < 0.05). Additionally, Phe deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 mRNA expression (P < 0.05). These results may be ascribed to the improvement in reactive oxygen species (ROS) levels in the fish gills (P < 0.05). Furthermore, the results indicated that Phe deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gills (P < 0.05). These effects were partly due to the downregulation of interleukin 10 (IL-10), transforming growth factor ß (TGF-ß) and inhibitor factor κBα (iκBα) and the upregulation of relative mRNA expression of interleukin 1ß (IL-1ß), interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α) and nuclear transcription factor-κB p65 (NF-κB p65) (P < 0.05). Taken together, the results showed that Phe deficiency impaired the structural integrity of fish gills by regulating the expression of tight junction proteins, cytokines, antioxidant enzymes, NF-κB p65, iκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gills.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, ß-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) ß1, TGF-ß2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1ß, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Deficiency of dietary niacin impaired gill immunity and antioxidant capacity, and changes its tight junction proteins via regulating NF-κB, TOR, Nrf2 and MLCK signaling pathways in young grass carp (Ctenopharyngodon idella).

To investigate the effects of dietary niacin on gill immunity, tight junction proteins, antioxidant system and related signaling molecules mRNA expression, young grass carp (Ctenopharyngodon idella) were fed six diets containing graded levels of niacin (3.95-55.01 mg/kg diet) for 8 weeks. The study indicated that niacin deficiency decreased lysozyme and acid phosphatase activities, and complement 3 content, and caused oxidative damage that might be partly due to the decreased copper, zinc superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase activities and reduced glutathione content in fish gills (P < 0.05). Moreover, the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and Hepcidin), anti-inflammatory cytokines (interleukin 10 and transforming growth factor ß1), tight junction proteins (Occludin, zonula occludens 1, Claudin-15 and -3), signaling molecules (inhibitor of κBα (IκBα), target of rapamycin (TOR), ribosomal protein S6 kinase 1 (S6K1) and NF-E2-related factor 2 (Nrf2)) and antioxidant enzymes were significantly decreased (P < 0.05) in niacin-deficient diet group. Conversely, the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1ß), signaling molecules (nuclear factor kappa B p65, IκB kinase α, IκB kinase ß, IκB kinase γ, Kelch-like-ECH-associated protein 1b, myosin light chain kinase and p38 mitogen-activated protein kinase (p38 MAPK) were significantly increased (P < 0.05) in fish gills fed niacin-deficient diet. Interestingly, the varying niacin levels of 3.95-55.01 mg/kg diet had no effect on the mRNA level of Kelch-like-ECH-associated protein 1a, Claudin-c and -12 in fish gills (P > 0.05). In conclusion, niacin deficiency decreased gill immunity, impaired gill antioxidant system, as well as regulated mRNA expression of gill tight junction proteins and related signaling molecules of fish.

Dietary choline regulates antibacterial activity, inflammatory response and barrier function in the gills of grass carp (Ctenopharyngodon idella).

An 8-week feeding trial was conducted to determine the effects of graded levels of choline (197-1795 mg/kg) on antibacterial properties, inflammatory status and barrier function in the gills of grass carp. The results showed that optimal dietary choline supplementation significantly improved lysozyme and acid phosphatase activities, complement component 3 (C3) content, and the liver expressed antimicrobial peptide 2 and Hepcidin mRNA levels in the gills of fish (P < 0.05). In addition, appropriate dietary choline significantly decreased the oxidative damage, which might be partly due to increase copper, zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) activities and increased glutathione content in the gills of fish (P < 0.05). Moreover, appropriate dietary choline significantly up-regulated the mRNA levels of interleukin 10 and transforming growth factor ß1, Zonula occludens 1, Occludin, Claudin-b, c, 3 and 12, inhibitor of κBα, target of rapamycin, Cu/Zn-SOD, CAT, GR, GPx, GST and NF-E2-related factor 2 in the gills of fish (P < 0.05). Conversely, appropriate dietary choline significantly down-regulated the mRNA levels of pro-inflammatory cytokines, tumor necrosis factor α, interleukin 8, interferon γ, interleukin 1ß, and related signaling factors, nuclear factor kappa B p65, IκB kinase ß, IκB kinase γ, myosin light chain kinase and Kelch-like-ECH-associated protein 1a (Keap1a) in the gills of fish (P < 0.05). However, choline did not have a significant effect on the mRNA levels of IκB kinase α, Claudin-15 and Keap1b in the gills of fish. Collectively, appropriate dietary choline levels improved gill antibacterial properties and relative gene expression levels of tight junction proteins, and decreased inflammatory status, as well as up-regulated the mRNA levels of related signaling molecules in the gills of fish. Based on gill C3 content and AHR activity, the dietary choline requirements for young grass carp (266.5-787.1 g) were estimated to be 1191.0 and 1555.0 mg/kg diet, respectively.

Modulation of immune response, physical barrier and related signaling factors in the gills of juvenile grass carp (Ctenopharyngodon idella) fed supplemented diet with phospholipids.

This study was conducted to investigate the effects of dietary phospholipids (PL) on the gill immune response and physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Compared with the control group, optimal PL supplementation increased (P < 0.05): (1) the lysozyme activity, acid phosphatase activity, complement component 3 (C3) content, liver expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 mRNA expression; (2) the relative mRNA expression of interleukin 10, transforming growth factor ß1, inhibitor factor κBα (IκBα) and target of rapamycin (TOR); (3) the activities of anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), copper/zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), glutathione content and mRNA levels of SOD1, CAT, GPx, GR and NF-E2-related factor 2 (Nrf2) genes; (4) the transcription abundance of occludin, claudin b, claudin c, claudin 12 and zonula occludens 1 genes. At the same time, appropriate PL supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1ß, nuclear factor κB p65 (NF-κB p65), IκB kinase ß (IKKß) and IκB kinase γ (IKKγ) mRNA expression; (2) malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) content and the relative mRNA expression of Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b; (3) the transcription abundance of myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) genes. In conclusion, the positive effect of PL on gill health is associated with the improvement of the immunity, antioxidant status and tight junction barrier of fish gills. Finally, based on ACP activity, C3 content, PC content and ASA activity in the gills, the optimal dietary PL level for juvenile grass carp (9.34-87.50 g) was estimated to be 3.62%, 4.30%, 3.91% and 3.86%, respectively.

Comparative tissue expression of American lobster (Homarus americanus) immune genes during bacterial and scuticociliate challenge.

The American lobster (Homarus americanus) fishery is the most economically significant fishery in Canada; although comparatively little is known about the lobsters' response to pathogenic challenge. This is the first study to investigate the expression of immune genes in tissues outside of the lobster hepatopancreas in response to challenges by the Gram-positive bacteria, Aerococcus viridans var. homari or the scuticociliate parasite, Anophryoides haemophila. The hepatopancreas has been regarded as the major humoral immune organ in crustaceans, but the contribution of other organs and tissues to the molecular immune response has largely been overlooked. This study used RT-qPCR to monitor the gene expression of several immune genes including three anti-lipopolysaccharide isoforms (ALF) Homame ALF-B1, Homame ALF-C1 and ALFHa-1, acute phase serum amyloid protein A (SAA), as well as thioredoxin and hexokinase, in antennal gland and gill tissues. Our findings indicate that the gene expression of the SAA and all ALF isoforms in the antennal gland and gill tissues increased in response to pathogenic challenge. However, there was differential expression of individual ALF isoforms that were dependent on both the tissue, and the pathogen used in the challenge. The gene expression changes of several immune genes were found to be higher in the antennal gland than have been previously reported for the hepatopancreas. This study demonstrates that increased immune gene expression from the gill and antennal gland over the course of pathogen induced disease contributes to the immune response of H. americanus.

Dietary pantothenic acid depressed the gill immune and physical barrier function via NF-κB, TOR, Nrf2, p38MAPK and MLCK signaling pathways in grass carp (Ctenopharyngodon idella).

This study explored the effects of pantothenic acid (PA) on the immune and physical barrier function, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella). The results indicated that compared with optimal PA supplementation, PA deficiency (1.31 mg/kg diet) decreased gill interleukin 10, transforming growth factor ß1, inhibitor of κBα (IκBα), eIF4E-binding protein 2, Claudin b and ZO-1 mRNA levels; anti-superoxide anion activity, and activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, glutathione reductase and NF-E2-related factor (P < 0.05). Additionally, PA deficiency and excess (75.08 mg/kg diet) decreased gill complement 3 and glutathione contents, lysozyme and acid phosphatase, anti-hydroxy radical, catalase and glutathione S-transferases activities, and liver-expression antimicrobial peptide 2, hepcidin, Claudin 3, Claudin c and Occludin mRNA levels (P < 0.05). Conversely, PA deficiency increased gill reactive oxygen species and protein carbonyl contents, and interferon γ2, interleukin 8, nuclear factor kappa B P65, Claudin 15a, Kelch-like ECH-associating protein 1a and Kelch-like ECH-associating protein 1b mRNA levels (P<0.05). Moreover, PA deficiency and excess increased gill malondialdehyde content, and tumor necrosis factor α, interleukin 1ß, IκB kinase α, IκB kinase ß, IκB kinase γ, target of rapamycin and ribosomal S6 protein kinase1 p38 mitogen-activated protein kinases and myosin light-chain kinase mRNA levels (P<0.05). In conclusion, PA deficiency decreased immune and physical barrier function, and regulated relative mRNA levels of signaling molecules in fish gill. Based on the quadratic regression analysis of gill lysozyme activity, the optimal PA levels in grass carp (253.44-745.25 g) were estimated to be 36.97 mg/kg diet.

Folic acid deficiency impairs the gill health status associated with the NF-κB, MLCK and Nrf2 signaling pathways in the gills of young grass carp (Ctenopharyngodon idella).

The aim of this study was to investigate the effect of dietary folic acid on fish growth, the immune and barrier functions of fish gills, and the potential mechanisms of these effects. Young grass carp (Ctenopharyngodon idella) were fed diets containing graded levels of folic acid at 0.10 (basal diet), 0.47, 1.03, 1.48, 1.88 and 3.12 mg kg(-1) diet for 8 weeks. The results showed that acid phosphatase and lysozyme activities and the complement component 3 content in fish gills decreased with folic acid deficiency (P < 0.05). Folic acid deficiency up-regulated liver-expressed antimicrobial peptide 1, interleukin 1ß, interleukin 8, tumor necrosis factor α, nuclear factor κB p65, IκB kinase α (IKK-α), IKK-ß and IKK-γ gene expression. Folic acid deficiency down-regulated interleukin 10, transforming growth factor ß, IκB and target of rapamycin gene expression in fish gills (P < 0.05). These results showed that limited folic acid decreased fish gill immune status. Furthermore, folic acid deficiency down-regulated claudin-b, claudin-c, claudin-3, occludin and zonula occludens 1 gene expression, whereas folic acid deficiency up-regulated claudin-12, claudin-15, myosin light chain kinase and p38 mitogen activated protein kinase gene expression in fish gills (P < 0.05). These results suggested that folic acid deficiency disrupted tight junction-mediated fish gill barrier function. Additionally, folic acid deficiency increased the content of reactive oxygen species, protein carbonyl and malondialdehyde (MDA); Mn superoxide dismutase activity and gene expression; and Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b gene expression (P < 0.05). Conversely, folic acid deficiency decreased Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione s-transferases and glutathione reductase activities and gene expression as well as NF-E2-related factor 2 gene expression in fish gills (P < 0.05). All of these results indicated that folic acid deficiency impaired fish gill health status via regulating gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, MLCK and Nrf2. Based on percent weight gain, LZ activity and MDA content in the gills, the dietary folic acid requirements for young grass carp were 1.60, 2.07 and 2.08 mg kg(-1), respectively.

A manganese superoxide dismutase (MnSOD) from ark shell, Scapharca broughtonii: Molecular characterization, expression and immune activity analysis.

Manganese superoxide dismutase (MnSOD) is one of the key members of the antioxidant defense enzyme family, however, data regarding to the immune function of MnSOD in mollusks still remain limited now. In this study, a full-length MnSOD cDNA was identified by rapid amplification of cDNA ends (RACE) method from cDNA library of ark shell Scapharca broughtonii (termed SbMnSOD). The cDNA contained an open reading frame (ORF) of 696 bp which encoded a polypeptide of 232 amino acids, a 5'-UTR with length of 32 bp and a 3'-UTR of 275 bp. Four putative amino acid residues (His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of SbMnSOD and the signature sequence (DVWEHAYY) also existed in SbMnSOD. The deduced amino acid sequence of SbMnSOD shared high homology to MnSOD from other species. All those data revealed that the SbMnSOD was a novel member of the MnSOD family. The mRNA expression profiles of SbMnSOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbMnSOD distributed in all the examined tissues. Importantly, Vibrio anguillarum challenge resulted in the increased expression of SbMnSOD mRNA with a regular change trend in all examined tissues, indicating SbMnSOD actively participated in the immune response process. What's more, further analysis on the antibacterial activity of the recombinant SbMnSOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria. The present results clearly suggested that SbMnSOD was an acute phase protein involved in the immune reaction in S. broughtonii.

A transcriptomic analysis of American lobster (Homarus americanus) immune response during infection with the bumper car parasite Anophryoides haemophila.

Anophryoides haemophila is an important protistan parasite of American lobster, Homarus americanus, as it has been found to infect lobsters in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of over 14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to elucidate molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several anti-lipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms was monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RT-qPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5-7weeks for ALFHa-1 and 10weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during Aerococcus viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health.

Morphology and pathology of the ectoparasitic copepod, Nicothoë astaci ('lobster louse') in the European lobster, Homarus gammarus.

Ectoparasitic copepods have been reported in a wide range of aquatic animals, including crustacean shellfish. However, with the exception of the salmon louse, Lepeophtheirus salmonis, our knowledge of such parasites in commercial species is rudimentary. The current study examines the morphology and pathology of the parasitic copepod, Nicothoë astaci (the 'lobster louse') in its host, the European lobster, Homarus gammarus. Lobsters were sampled from waters surrounding Lundy Island (Bristol Channel, UK) and all individuals collected were found to harbour female adult N. astaci in their gills, with a mean of 47·3 parasites/lobster. The majority of N. astaci were found in the basal region of pleurobranch gills. The parasite was found to attach to gill filaments via its oral sucker, maxillae and maxillipeds, and to feed on host haemolymph (blood) through a funnel-like feeding channel. It caused varying degrees of damage to the host gill, including occlusion of gill filaments and disruption to the vascular system in the central axis. Although there was evidence of extensive host response (haemocytic infiltration) to the parasite, it was displaced from the parasite attachment site and thus was observed in the central gill axis below. The region of gill filament immediately underlying the parasite feeding channel was devoid of such activity suggesting that the parasite interferes with the cellular defence and haemostatic mechanisms of the lobster in order to maintain invasion of the host.