Postprandial plasma amino acid and appetite responses with ingestion of a novel salmon-derived protein peptide in healthy young adults.

This study assessed postprandial plasma aminoacidemia, glycemia, insulinemia and appetite responses to ingestion of a novel salmon-derived protein peptide (Salmon PP) compared with milk protein isolate (Milk PI). In a randomised, participant-blind crossover design, eleven healthy adults (M = 5, F = 6; mean ± sd age: 22 ± 3 years; BMI: 24 ± 3 kg/m2) ingested 0·3 g/kg/body mass of Salmon PP or Milk PI. Arterialised blood samples were collected whilst fasted and over a 240-min postprandial period. Appetite sensations were measured via visual analogue scales. An ad libitum buffet-style test meal was administered after each trial. The incremental AUC (iAUC) plasma essential amino acid (EAA) response was similar between Salmon PP and Milk PI. The iAUC plasma leucine response was significantly greater following Milk PI ingestion (P < 0·001), whereas temporal and iAUC plasma total amino acid (P = 0·001), non-essential amino acid (P = 0·002), glycine (P = 0·0025) and hydroxyproline (P < 0·001) responses were greater following Salmon PP ingestion. Plasma insulin increased similarly above post-absorptive values following Salmon PP and Milk PI ingestion, whilst plasma glucose was largely unaltered. Indices of appetite were similarly altered following Salmon PP and Milk PI ingestion, and total energy and macronutrient intake during the ad libitum meal was similar between Salmon PP and Milk PI. The postprandial plasma EAA, glycine, proline and hydroxyproline response to Salmon PP ingestion suggest this novel protein source could support muscle and possibly connective tissue adaptive remodelling, which warrants further investigation, particularly as the plasma leucine response to Salmon PP ingestion was inferior to Milk PI.

Serum proteinogram of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) as a new useful approach for detecting loss of haemostasis.

Proteinograms, a semiquantitative analytical method that separates proteins into multiple bands, have not been explored in teleosts for diagnostic or prognostic purposes. This study aimed to establish reference values for proteinograms in the serum of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), two important farmed fish species in the Mediterranean region. Serum proteins were studied using SDS-PAGE, electropherogram, and HPLC-mass spectrometry. SDS-PAGE analysis revealed four major bands of proteins around 11, 25, 70, and 100 kDa in the serum of gilthead seabream and European sea bass. Electropherogram results showed that a protein with a molecular weight of 76.8 kDa was the most abundant protein in the serum of gilthead seabream, while a peak of 75.5 kDa was the most abundant in European sea bass. HPLC-mass spectrometry detected 87 proteins and 119 proteins in the serum of gilthead seabream and European sea bass, respectively, including α1-globulins, α2-globulins, ß-globulins, and γ-globulins. Notably, the albumin sequence was not detected in either of the two species. These results help to characterize the serum protein profile and to establish reference proteinograms for these two fish species. They also provide a basis for the development of novel approaches for the rapid detection of loss of haemostasis due to stress, health disorders or disease in farmed fish.

Short time-series expression transcriptome data reveal the gene expression patterns and potential biomarkers of blood infection with LPS and poly (I:C) in Mandarin fish (Siniperca chuatsi).

Blood transcriptomics has emerged as a vital tool for tracking the immune system and supporting disease diagnosis, prognosis, treatment, and research. The present study was conducted to analyze the gene expression profile and potential biomarker candidates using the whole blood of mandarin fish (Siniperca chuatsi) infected with LPS or poly (I:C) at 0 h, 3 h, 6 h, and 12 h. Our data suggest that 310 shared differentially expressed genes (DEGs) were identified among each comparison group after LPS infection, and 137 shared DEGs were identified after poly (I:C) infection. A total of 62 shared DEGs were differentially expressed in all compared groups after LPS or poly (I:C) infection. Pathways analysis for DEGs in all different compared groups showed that cytokine-cytokine receptor interaction was the most enrichment pathway. The expression levels of genes C-X-C chemokine receptor type 2-like (cxcr2), chemokine (C-C motif) receptor 9a (ccr9a), chemokine (C-C motif) receptor 9b (ccr9b), chemokine (C-X-C motif) receptor 4b (cxcr4b), and interleukin 10 receptor alpha (il10ra) were significantly different in all compared groups and most enriched in cytokine-cytokine receptor interaction pathway. The protein-protein interactions analysis among all shared DEGs showed that cxcr4 was the hub gene with the highest degree. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring mandarin fish diseases.

Evaluation of circulating insulin-like growth factor (IGF)-I and IGF-binding proteins as growth indices in rainbow trout (Oncorhynchus mykiss).

Circulating insulin-like growth factor (IGF)-I has been proposed as a growth index in several teleosts, including salmonids, and its level in circulation is stabilized by multiple IGF-binding proteins (IGFBPs). Three IGFBPs, IGFBP-2b, -1a, and -1b, are consistently detected in salmonid blood and are suggested to be indices of positive or negative growth, although their applicability to rainbow trout (Oncorhynchus mykiss) is unclear. The present study examined the usefulness of IGFBPs along with IGF-I as a physiological indicator of growth rate in rainbow trout through a rearing experiment. Two groups of underyearling rainbow trout were pit-tagged and either fed or fasted for 33 days. A third group was fasted for 22 days, followed by refeeding for 11 days. Serum IGF-I levels were reduced after fasting for 22 days, but refeeding did not retore its levels to those of the fed control. Nevertheless, there was a positive relationship between serum IGF-I levels and individual growth rates over 33 days of experimentation, confirming its validity as a growth index. Ligand blotting using labeled human IGF-I revealed two IGFBP bands at 43 and 32 kDa, which corresponded to IGFBP-2b and an unidentified form, respectively. In contrast, bands corresponding to IGFBP-1a and -1b, which usually increase after fasting, were hardly detected, even in the fasted fish. The responses of circulating IGFBP-2b to fasting and refeeding were similar to those of circulating IGF-I and positively correlated with growth rate and IGF-I levels. The intensity of the serum 32-kDa IGFBP band was higher in constantly fed fish than in the fasted fish; however, its correlation with growth rate was weaker than those of IGF-I and IGFBP-2b. The present study shows that IGF-I and IGFBP-2b can be used as growth indices for rainbow trout. In contrast, circulating IGFBP-1a and -1b may not serve as negative growth indices in rainbow trout under regular aquaculture conditions because they are rarely detected by ligand blotting or respond to fasting/refeeding.

Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation.

The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May.

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing.

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cµ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cµ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cµ locus has a Cµ1-Cµ2-Cµ3-Cµ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cµ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Molecular evolution of kalliklectin in teleost and identification of the novel type with eight apple domains in channel catfish, Ictalurus punctatus.

Kalliklectin is a unique fish-specific lectin that demonstrates sequence similarity to mammalian plasma kallikrein and coagulation factor XI, which are not lectins but proteases. Reported fish kalliklectins and these mammalian proteases comprise four characteristic "apple domains" (APDs). Bioinformatics analysis revealed that Siluriformes species possess anomalous kalliklectins comprising 6 to 16 APDs. Complementary DNA cloning showed that the full-length nucleotide sequence of Ictalurus punctatus consists of 2240 bp that encode 720 amino acid residues to produce a mature protein with a putative 18 amino acid N-terminus peptide sequence. This protein has a predicted molecular mass of 83,417.23 Da. Reverse transcription-polymerase chain reaction (RT-PCR) showed that this lectin gene expresses in the liver but not in any other tissues, including the mucosal tissues. This differential expression pattern makes this lectin unique compared to other lectins described in previous studies. We successfully detected an 85-kDa protein in the serum using western blotting analysis, suggesting that this lectin protein is produced by the liver and secreted into the bloodstream. We characterized a novel cDNA sequence encoding a new type of kalliklectin with eight APDs isolated from channel catfish, I. punctatus. Based on phylogenetic analysis, we speculated that there was a duplication of the third and fourth APD set in a common Siluriformes ancestor at some point after its separation from the common teleost ancestor and that these duplications then underwent independent repeats in different lineages resulting in the generation of the [APD1]-[APD2]-{[APD3]-[APD-4]} × n structure in modern catfishes.

Effects of growth hormone and cortisol administration on plasma insulin-like growth factor binding proteins in juveniles of three subspecies of masu salmon (Oncorhynchus masou).

In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semi-quantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, -1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-1b levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles-Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways.

Extracellular vesicles (EVs) are lipid bilayer vesicles which are released from cells and play multifaceted roles in cellular communication in health and disease. EVs can be isolated from various body fluids, including serum and plasma, and are usable biomarkers as they can inform health status. Studies on EVs are an emerging research field in teleost fish, with accumulating evidence for important functions in immunity and homeostasis, but remain to be characterised in most fish species, including halibut. Protein deimination is a post-translational modification caused by a conserved family of enzymes, named peptidylarginine deiminases (PADs), and results in changes in protein folding and function via conversion of arginine to citrulline in target proteins. Protein deimination has been recently described in halibut ontogeny and halibut serum. Neither EV profiles, nor total protein or deiminated protein EV cargos have yet been assessed in halibut and are reported in the current study. Halibut serum EVs showed a poly-dispersed population in the size range of 50-600 nm, with modal size of EVs falling at 138 nm, and morphology was further confirmed by transmission electron microscopy. The assessment of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis showed that deimination hits are involved in a range of gene regulatory, immune, metabolic and developmental processes. The same was found for total EV protein cargo, although a far wider range of pathways was found than for deimination hits only. The expression of complement component C3 and C4, as well as pentraxin-like protein, which were identified by proteomic analysis, was further verified in EVs by western blotting. This showed that C3 is exported in EVs at higher levels than C4 and deiminated C3 was furthermore confirmed to be at high levels in the deimination-enriched EV fractions, while, in comparison, C4 showed very low detection in deimination-enriched EV fractions. Pentraxin was exported in EVs, but not detected in the deimination-enriched fractions. Our findings provide novel insights into EV-mediated communication in halibut serum, via transport of protein cargo, including post-translationally deiminated proteins.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Stocking density alters growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) in in-pond raceway system.

The effects of stocking density on growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) reared in nine in-pond raceway systems (IPRS, 22.0 m × 5.0 m × 2.0 m) were studied. M. salmoides with initial an body weight of 8.25 ± 0.51 g and body length of 6.99 ± 0.44 cm were reared at an initial stocking density of 90.91 ind./m3 (low stocking density, LSD), 113.63 ind./m3 (middle stocking density, MSD), and 136.36 ind./m3 (high stocking density, HSD) with triplication. After 300 days of culture, MSD recorded the highest final body weight, weight gain, specific growth rate, and yield, but the food conversion ratio in MSD was the lowest. The viscerosomatic index in LSD was significantly higher than other groups. The fish serum reared at HSD showed significantly lower total protein, higher total cholesterol, triglyceride, total bilirubin, glucose content, alanine transaminase, and aspartate transaminase activity. Significantly lower intestinal amylase, lipase, trypsin activities, hepatic superoxide dismutase (SOD) and catalase (CAT) activities, and higher malondialdehyde content were detected in HSD compared to others. The content of crude lipid, saturated fatty acid decreased, and total essential amino acid, delicious amino acid, and polyunsaturated fatty acid increased in muscle with stocking density increase. No significant difference was observed in muscle texture. Profitability analysis indicated the benefit-to-cost ratio varied between 1.10 and 1.68, of which MSD was significantly higher than others. The optimal stocking density for M. salmoides should be 113.63 ind./m3 in an IPRS farm.

The blood and mRNA levels of antioxidant-related factors in common carp (Cyprinus carpio) fed p-Coumaric acid.

The natural antioxidants are well known for their antioxidative activity without side effects when compared to antibiotics. Hence, the present study aimed at evaluating p-Coumaric acid as an antioxidant additive on the blood and mRNA levels of antioxidant-related factors in common carp (Cyprinus carpio). Fish fed the basal diet supplemented with p-Coumaric at 0, 0.5, 1, and 1.5 g/kg for 56 days, then the serum, intestine, and liver samples were collected. The growth performance of fish fed with CA showed significantly (P < 0.05) improved FW, WG, and SGR compared to those of the control one. However, the feed conversion ratio was significantly (P < 0.05) reduced in fish fed 1 and 1.5 g/kg diet levels. SOD was not significantly differed among the groups fed with varied p-Coumaric acid (P > 0.05). Serum GPX and TAC were enhanced considerably by p-Coumaric acid regarding the control with the highest being in fish fed 1.5 g/kg diet (P < 0.05). Serum CAT was more elevated in fish provided p-Coumaric acid at 1 or 1.5 g/kg than the control while fish fed 0.5 g/kg did not display significant changes. MDA level significantly decreased by all p-Coumaric acid groups compared to the control one, and the lowest level was observed in 1.5 g/kg (P < 0.05). The mRNA level of CAT was significantly upregulated in the liver by p-Coumaric acid at 1 or 1.5 g/kg (P < 0.05), while the intestine CAT did not influence by p-Coumaric acid (P > 0.05). The measured SOD in the liver and intestine samples revealed no changes in common carp fed p-Coumaric acid (P > 0.05). GPX was significantly upregulated in the intestine by p-Coumaric acid at 1 or 1.5 g/kg (P < 0.05), whereas the liver GPX was upregulated by p-Coumaric acid at 1.5 g/kg. The mRNA level of the GST gene in the intestine of common carp was upregulated by p-Coumaric acid at 1.5 g/kg, whereas the liver displayed upregulated GST in fish fed 1 g/kg diet. The present study approved the application of p-Coumaric acid as a natural antioxidant for friendly, sustainable aquaculture.

Assesment of hematotoxic, oxidative and genotoxic damage potentials of fipronil in rainbow trout Oncorhynchus mykiss, Walbaum.

In this study, changes in the blood tissue of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) caused by Fipronil (FP) insecticide were investigated using different biomarkers (Hematology parameters, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), paraoxonase (PON), arylesterase (ARE), myeleperoxidase (MPO), micronucleus (MN), 8-hydroxy-2-deoxyguanosine (8-OHdG)) level and caspase-3 activity. Statistically significant alterations in hematology parameters occurred with FP effect. In blood tissue, dose-dependent inhibition was determined in SOD-CAT-GPX-PON and ARE enzyme activities, but MDA and MPO were induced statistically significant. The results of MN assay were compared with the control group and it was obtained that genotoxicity of different dose groups was similar. The level of 8-OHdG and the activity and caspase-3 examined in blood tissue was increased depending on the dose. It was determined with different biomarkers that this insecticide caused physiological stress changes in the tissues examined.

Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research.

BACKGROUND: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. RESULTS: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. CONCLUSIONS: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

Intact rather than total circulating insulin-like growth factor binding protein-1a is a negative indicator of growth in masu salmon.

Insulin-like growth factor binding protein (IGFBP)-1a is one of three major circulating forms in salmon and induced under catabolic conditions. However, there is currently no immunoassay available for this form because of a lack of standard and specific antibodies. We developed a time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1a using recombinant protein for labeling, an assay standard, and production of antiserum. The TR-FIA had a low cross-reactivity (3.6%) with IGFBP-1b, another major form in the circulation. Fasting for 4 wk had no effect on serum immunoreactive (total) IGFBP-1a levels in yearling masu salmon, whereas 6-wk fasting significantly increased it. There was a significant, but weak, negative relationship between serum total IGFBP-1a level and individual growth rate (r2 = 0.12, P = 0.01). We next developed a ligand immuno-functional assay (LIFA) using europium-labeled IGF-I to quantify intact IGFBP-1a. In contrast to total IGFBP-1a, serum intact IGFBP-1a levels increased after 4 wk of fasting, and refeeding for 2 wk restored it to levels similar to those of the fed control. Serum intact IGFBP-1a levels showed a significant negative correlation with individual growth rate (r2 = 0.52, P < 0.001), which was as good as that of IGFBP-1b. Our findings using newly developed TR-FIA and LIFA suggest that regulation of intact IGFBP-1a levels has an important effect on growth in salmon and that intact IGFBP-1a is a negative index of salmon growth.

Quantitative proteomic analysis in serum of Takifugu rubripes infected with Cryptocaryon irritans.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p < 0.05, fold-change threshold ≥ 2), including 21 up-regulated and 44 down-regulated. Combined with STRING-based functional analysis, we ultimately obtained eight proteins including glucokinase-like, integrin beta-1-like isoform X2, H4, H2A.V, histone H1-like, histone H2AX-like, histone H2B 1/2-like and myosin-9 isoform X1, which could be considered as potential biomarkers for T. rubripes immune responses. Eight proteins that were selected to validate significant differentially expressed genes at the proteomic level were consistent with qPCR at the transcriptomic level. The proteins identified in our work may serve as candidates for elucidating the molecular mechanism of cryptocaryonosis in T. rubripes. Our collective findings could provide new insights into searching for disease-specific targets and biomarkers, which may be effective indicators of C. irritans infection in T. rubripes.

Immediate increase of plasma protein complement C3 in response to an acute stressor.

Whereas chronic stress has immunosuppressive effects, the more immediate immunologic consequences of acute stressors are less known. We postulated that, as part of their 'fight or flight' response, rainbow trout would rapidly increase the efficacy of their natural immune system by means of increased concentrations of crucial plasma proteins. Plasma samples were taken from resting fish and from fish 5, 10 or 20 min after initiation of a stressful regime. Using crossed immunoelectrophoresis, we documented increases in concentrations of complement C3 and 3 other proteins within 5 min of initiation of stress. The concentration of C3 nearly doubled within 10 min of initiation of stress and had returned to near resting level by 20 min. This rapid kinetics preclude dependence on gene activation, the basis of the acute phase response. Potentiation of natural immunity, which can reasonably be expected to be selectively advantageous during or immediately after acute stressors may be one result of this increase.

Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: a randomised, double blind study.

PURPOSE: To examine whether supplementation with low doses of fish or milk proteins would affect glucose regulation and circulating lipid concentrations in overweight healthy adults. METHODS: Ninety-three overweight adults were assigned to receive 2.5 g protein/day from herring (HER), salmon (SAL), cod (COD) or milk (CAS, a casein-whey mixture as positive control) as tablets for 8 weeks. RESULTS: Seventy-seven participants were included in the analyses. HER and SAL did not affect glucose and insulin concentrations. COD significantly reduced within-group changes in 90 and 120 min postprandial glucose concentrations but changes were not different from HER and SAL groups. CAS supplementation significantly reduced the area under the curve for glucose concentrations (- 7%), especially when compared to SAL group, and reduced postprandial insulin c-peptide concentration (- 23%). Reductions in acetoacetate (- 24%) and ß-hydroxybutyrate (- 29%) serum concentrations in HER group were more prominent compared to SAL and COD groups, with no differences between fish protein groups for α-hydroxybutyrate. Serum concentrations of α-hydroxybutyrate (- 23%), acetoacetate (- 39%) and ß-hydroxybutyrate (- 40%) were significantly reduced within CAS group, and the decreases were significantly more pronounced when compared to SAL group. Serum lipid concentrations were not altered in any of the intervention groups. CONCLUSION: Findings indicate that 2.5 g/day of proteins from fish or milk may be sufficient to improve glucose regulation in overweight adults. The effects were most pronounced after supplementation with proteins from cod, herring and milk, whereas salmon protein did not affect any of the measurements related to glucose regulation. CLINICAL TRAIL REGISTRATION: This trial was registered at clinicaltrials.gov as NCT01641055.

Effect of photoperiod and temperature on indicators of immunity and wellbeing of endangered golden mahseer (Tor putitora) broodstock.

Recently, we reported the possibility of successfully inducing captive maturation and spawning in golden mahseer through photothermal manipulation. Subsequently, we felt that it was imperative to understand the impact of these environmental manipulations on immunity, stress response, antioxidant potential, and general well-being of adult mahseer to develop a healthy broodstock. For this purpose, two experiments were carried out with changes in photoperiod (experiment I) and temperature (experiment II). In experiment I, random groups of adult female and male Tor putitora were subjected to three photoperiods (8L:16D, 12L:12D and 16L:8D) for 100 days. Decreasing levels of plasma melatonin with increasing photoperiod confirmed the physiological significance of different light-dark conditions in mahseer brooders. In terms of stress, plasma cortisol levels showed a linear increase with decreasing light duration in both males and females. Similarly, the level of thiobarbituric acid reactive substances was also significantly higher in males kept at 8L:16D. Plasma concentration of total immunoglobulins was found reduced in female brooders at 8L:16D, but this was not evident in males. In females, total antioxidants were found significantly elevated at 12L:12D. On the contrary, superoxide dismutase activity was lower at 12L:12D in females. The photoperiod has substantially influenced the plasma total protein and albumin levels in males. In experiment II, random groups of adult T. putitora were reared at ambient (21.2 ± 1.4 °C) or elevated temperature (23.7 ± 1.3 °C) groups for 121 days. The higher temperature was found to significantly decrease lysozyme, myeloperoxidase, and anti-protease activities in female mahseer brooders. However, total immunoglobulin levels were reduced significantly at elevated temperature both in males and females. No other temperature-related significant changes were observed in antioxidant potential, anti-oxidative enzymes or well-being related indices.