Developmental changes in digestive enzyme activity in American shad, Alosa sapidissima, during early ontogeny.

In order to assess the digestive physiological capacity of the American shad Alosa sapidissima and to establish feeding protocols that match larval nutritional requirements, we investigated the ontogenesis of digestive enzymes (trypsin, amylase, lipase, pepsin, alkaline phosphatase, and leucine aminopeptidase) in larvae, from hatching to 45 days after hatching (DAH). We found that all of the target enzymes were present at hatching, except pepsin, which indicated an initial ability to digest nutrients and precocious digestive system development. Trypsin rapidly increased to a maximum at 14 DAH. Amylase sharply increased until 10 DAH and exhibited a second increase at 33 DAH, which coincided with the introduction of microdiet at 30 DAH, thereby suggesting that the increase was associated with the microdiet carbohydrate content. Lipase increased until 14 DAH, decreased until 27 DAH, and then increased until 45 DAH. Pepsin was first detected at 27 DAH and then sharply increased until 45 DAH, which suggested the formation of a functional stomach. Both alkaline phosphatase and leucine aminopeptidase markedly increased until 18 DAH, which indicated intestinal maturation. According to our results, we conclude that American shad larvae possess the functional digestive system before mouth opening, and the significant increases in lipase, amylase, pepsin, and intestinal enzyme activities between 27 and 33 DAH suggest that larvae can be successfully weaned onto microdiets around this age.

Effects of stocking density on antioxidant status, metabolism and immune response in juvenile turbot (Scophthalmus maximus).

This study was designed to evaluate the physiological and immune responses of juvenile turbot to stocking density. Turbot (average weight 185.4g) were reared for 120days in a land based recirculating aquaculture system (RAS) under three stocking densities: low density (LD, ~9.3-26.1kg/m2, initial to final density), medium density (MD, ~13.6-38.2kg/m2) and high density (HD, ~19.1-52.3kg/m2). Fish were sampled at days 0, 40, 80 and 120 to obtain growth parameters and liver tissues. No significant difference was detected in growth, biochemical parameters and gene expression among the three densities until at the final sampling (day 120). At the end of this trial, fish reared in HD group showed lower specific growth rate (SGR) and mean weight than those reared in LD and MD groups. Similarly, oxidative stress and metabolism analyses represented that antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione (GSH)) and metabolic enzymes (glycerol-3-phosphate dehydrogenase (G3PDH) and glucose-6-phosphate dehydrogenase (G6PDH)) clearly reduced in the liver of turbot reared in HD group. The gene expression data showed that glutathione S-transferase (GST), cytochrome P450 1A (CYP1A), heat shock protein 70 (HSP 70) and metallothionein (MT) mRNA levels were significantly up-regulated, and lysozyme (LYS) and hepcidin (HAMP) mRNA levels were significantly down-regulated in HD group on day 120. Overall, our results indicate that overly high stocking density might block the activities of metabolic and antioxidant enzymes, and cause physiological stress and immunosuppression in turbot.

Molecular characterization and expression analysis of AMPK α subunit isoform genes from Scophthalmus maximus responding to salinity stress.

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722 bp) and α2 (1674 bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na+, K+-ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.

Stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities.

Fish skin and its mucus provide the first line of defense against chemical, physical and biological stressors, but little is known about the role of skin and its mucus in immune response to crowding stress. In the present study, we investigated the stress and immune responses in skin of turbot (Scophthalmus maximus) under different stocking densities. Turbot (average weight 185.4 g) were reared for 120 days under three densities: low density (LD), medium density (MD), and high density (HD). After 120 days, fish were weighed and sampled to obtain blood, mucus and skin tissues which were used for analyses of biochemical parameters and genes expression. The results showed HD treatment significantly suppressed growth and enhanced plasma cortisol and glucose levels (P < 0.05). In mucus, the activities of lysozyme (LZM), alkaline phosphatase (ALP) and esterase in HD treatment were lower than LD and MD treatments (P < 0.05) In skin, HD treatment resulted in up-regulation in malondialdehyde (MDA) formation and heat shock protein 70 (HSP 70) mRNA level, and down-regulation in activity of superoxide dismutase (SOD) and the transcriptions of glutathione-s-transferase (GST), interleukin-1ß (IL-1ß), tumor necrosis factor -α (TNF-α), insulin-like growth factor- (IGF-) and LZM (P < 0.05). Overall, the data suggested that overly high stocking density was a stressor which caused an immunosuppression in skin of turbot. Moreover, this information would help to understand the skin immunity and their relation with stress and disease in fish.

The physiological performance and immune response of juvenile turbot (Scophthalmus maximus) to nitrite exposure.

Nitrite (NO(2-)) is the most common toxic nitrogenous compound in aquatic environment. The aim of the present study was to investigate the effects of nitrite physiological performance and immune response of turbot. Fish were exposed to 0, 0.02, 0.08, 0.4 and 0.8 mM nitrite for 96 h. After 0, 24, 48 and 96 h of exposure, blood were collected to measure the levels of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), alkaline phosphatase (ALP), total protein (TP), albumin (Alb), complement C3 (C3), complement C4 (C4), immunoglobulin M (IgM) and lysozyme (LYS); gill samples were taken to analyze mRNA levels of LYS, heat shock protein 70 (HSP 70), heat shock protein 90 (HSP 90), metallothionein (MT), toll-like receptor 3 (TLR-3), tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and insulin-like growth factor I (IGF-I). The results showed that nitrite (0.4 and/or 0.8mM) significantly increased the levels of GPT, GOT, ALP, C3 and C4, reduced the levels of IgM and LYS, up-regulated the gene expressions of HSP 70, HSP 90, MT, TLR-3, TNF-α and IL-1ß, and down-regulated the gene expressions of LYS and IGF-1 after 48 and 96 h of exposure. Based on the results, it can be concluded that high level nitrite exposure results in dysfunction of the blood physiology and immunity in turbot. Further, this study will be helpful to understand the mechanism of aquatic toxicology induced by nitrite in marine fish.

Effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus).

Nitrite (NO2(-)) is commonly present as contaminant in aquatic environment and toxic to aquatic organisms. In the present study, we investigated the effects of nitrite exposure on haematological parameters, oxidative stress and apoptosis in juvenile turbot (Scophthalmus maximus). Fish were exposed to various concentrations of nitrite (0, 0.02, 0.08, 0.4 and 0.8mM) for 96 h. Fish blood and gills were collected to assay haematological parameters, oxidative stress and expression of genes after 0, 24, 48 and 96 h of exposure. In blood, the data showed that the levels of methemoglobin (MetHb), triglyceride (TG), potassium (K(+)), cortisol, heat shock protein 70 (HSP70) and glucose significantly increased in treatments with higher concentrations of nitrite (0.4 and/or 0.8mM) after 48 and 96 h, while the levels of haemoglobin (Hb) and sodium (Na(+)) significantly decreased in these treatments. In gills, nitrite (0.4 and/or 0.8mM) apparently reduced the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH), increased the formation of malondialdehyde (MDA), up-regulated the mRNA levels of c-jun amino-terminal kinase (JUK1), p53, caspase-3, caspase-7 and caspase-9 after 48 and 96 h of exposure. The results suggested caspase-dependent and JUK signaling pathways played important roles in nitrite-induced apoptosis in fish. Further, this study provides new insights into how nitrite affects the physiological responses and apoptosis in a marine fish.

[Progress in study on the skin mucus lectin in fish].

Since water is a perfect medium for both bacteria and parasitic microbes, fish skin is constantly exposed to pathogen attacks. It is generally believed that skin mucus serves a mechanical as well as biochemical barrier. Lectins, an important part of the mucus, are carbohydrate-binding proteins that are neither antibodies nor enzymes, yet play important roles in innate and adaptive immunity. Based on the structure of the carbohydrate recognition domain (CRD) and their function, fish mucus lectins are classified as four types. Recent research has shed light on the structural diversity and functions in innate immunity of mucus lectins. Here, we reviewed recent research progress on the classification, biological properties and functions of fish mucus lectins. Analyses on other fish species are therefore important in clarifying lectin diversity and their functions in skin mucus.

Characterization of RAG1 and IgM (mu chain) marking development of the immune system in red-spotted grouper (Epinephelus akaara).

In vertebrates, lymphoid-specific recombinase protein encoded by recombination-activating genes (RAG1/2) plays a key role in V(D)J recombination of the T-cell receptor and B-cell receptor. In this study, both RAG1 and the immunoglobulin M (IgM) mu chain were cloned to characterize their potential role in the immune defense at developmental stages of red-spotted grouper, Epinephelus akaara. The open reading frame (ORF) of E. akaara RAG1 included 2778 nucleotide residues encoding a putative protein of 925 amino acids, while the ORF of the IgM mu chain had 1734 nucleotide residues encoding 578 amino acids including variable (VH) and constant (CH1-CH2-CH3-CH4) regions. E. akaara RAG1 was composed of a zinc-binding dimerization domain (ZDD) with a RING finger and zinc finger A (ZFA) in the non-core region and a nonamer-binding region (NBR), with a zinc finger B (ZFB), the central and C-terminal domains in the core region. Tridimensional models of the ZDD and NBR of E. akaara RAG1 were constructed for the first time in fishes, while a 3D model of the E. akaara IgM mu chain was also clarified. The RAG1 mRNA was only detected in the thymus and kidney of 4-month and 1.5-year old groupers using qPCR, and the RAG1 protein was confirmed using western blotting and immunohistochemistry. The IgM mu mRNA was examined in most tissues except the gonad. RAG1 and IgM mu gene expression were observed at 15 dph (days post-hatching) and 23 dph respectively, and increased to a higher level at 37 dph. In addition, this was the first time that the morphology of the E. akaara thymus was characterized. The oval-shaped thymus of 4-month old fish was clearly seen and there were amounts of T lymphocytes present. The results suggested that the immune action of E. akaara was likely to start to develop around 15 dph to 29 dph. The transcript level of the RAG1 gene and the number of lymphocytes in the thymus between 4-month and 1.5-year old groupers indicated that age-related thymic atrophy also occurs in fishes. The similar functional structures of RAG1 and IgM protein between fish and mammals indicated that teleost species share a similar mechanism of V(D)J recombination with higher vertebrates.