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Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The risk of tuna adulteration is high driven by economic benefits. The authenticity of tuna is required to protect both consumers and tuna stocks. Given this, the study is designed to identify species-specific peptides for distinguishing three commercial tropical tuna species. The peptides derived from trypsin digestion were separated and detected using ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) in data-dependent acquisition (DDA) mode. Venn analysis showed that there were differences in peptide composition among the three tested tuna species. The biological specificity screening through the National Center for Biotechnology Information's Basic Local Alignment Search Tool (NCBI BLAST) revealed that 93 peptides could serve as potential species-specific peptides. Finally, the detection specificity of species-specific peptides of raw meats and processed products was carried out by multiple reaction monitoring (MRM) mode based on a Q-Trap mass spectrometer. The results showed that three, one and two peptides of Katsuwonus pelamis, Thunnus obesus and Thunnus albacores, respectively could serve as species-specific peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The need to fully exploit fishing resources due to increasing production and consequent waste generation requires research to promote the sustainability of the fishing industry. Fish waste from the industry is responsible for relevant environmental contamination. However, these raw materials contain high amounts of collagen and other biomolecules, being attractive due to their industrial and biotechnological applicability. Thus, to reduce the waste from pirarucu (Arapaima gigas) processing, this study aimed to obtain collagen from pirarucu skin tissue. The extraction process used 0.05 M sodium hydroxide, 10% butyl alcohol, and 0.5 M acetic acid, with extraction temperature of 20°C. The obtained yield was 27.8%, and through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), it was determined that the collagen obtained was type I. This study showed that collagen solubility was highest at pH 3 and the lowest solubility was at concentrations of 3% sodium chloride. The denaturation temperature of collagen was 38.1°C, and its intact molecular structure was observed using the Fourier transform infrared spectrophotometry technique with an absorption radius of 1. The results showed that it was possible to obtain collagen from pirarucu skin at 20°C, which has the typical characteristics of commercial type I collagen. In conclusion, the procedures used may be considered to be an interesting alternative for collagen extraction, a new product obtained from the processing of fish waste.

In this work, the effects of different QPE addition on the freeze-thaw (F-T) stability of fish myofibrillar protein (MP) gels were revealed. During freezing process, QPE decreased the freezing point of MP gels and shortened the time to pass through the maximum-ice-crystal-formation zone. The occurrence of thermal hysteresis effect led to the formation of small ice crystals and alleviated the damage to MP gel network. The incorporation of 7.5% QPE also reduced the free water amount to 19.23% and improved the water holding capacity of MP gels. Furthermore, the incorporation of QPE decreased the carbonyl content of MP gels after F-T cycles and delayed the protein oxidation. Meanwhile, QPE addition maintained the stability of the tertiary structure of MP gels via stabilizing the microenvironment of tyrosine and tryptophan. Overall, QPE shows the potential as a new cryoprotectant to improve the F-T stability of MP gel products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish., (© 2021 John Wiley & Sons Ltd.)

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. PRACTICAL APPLICATION: Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with >90% protein and peptide content, which is suitable for inclusion in foods., (© 2021 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

This study investigated how metabolite analysis can explain differences in tissue composition and size in fish from different habitats. We, therefore, studied Nile tilapia (Oreochromis niloticus) from three Ethiopian lakes (Gilgel Gibe, Ziway, and Langano) using dried bloodspot (DBS) analysis of carnitine esters and free amino acids. A total of sixty (N = 60) Nile tilapia samples were collected comprising twenty (n = 20) fish from each lake. The proximate composition of the targeted tissues (muscle, skin, gill, gut, and liver) were analyzed. The DBS samples were analyzed for acylcarnitine and free amino acid profiles using quantitative electrospray tandem mass spectrometry. Metabolite ratios were calculated from relevant biochemical pathways that could identify relative changes in nutrient metabolism. The mean weight of Nile tilapia sampled from each lake showed weight variation among the lakes, fish from Lake Ziway were largest (178 g), followed by Gilgel Gibe reservoir (134 g) and Lake Langano (118 g). Fish from Gilgel Gibe showed significantly higher fat composition in all tissues (P < 0.05) except the liver in which no significant variation was observed. The source of fish affected the tissue fat composition. Marked differences were observed in Nile tilapia metabolic activity between the lakes. For instance, the lower body weight and condition of the fish in Lake Langano coincided with several metabolite ratios pointing to a low flow of glucogenic substrate to the citric acid cycle. The low propionyl to acetylcarnitine ratio (C3:C2) in Gilgel Gibe fish is indicating that more of the available acetyl CoA is not led into the citric acid cycle, but instead will be used for fat synthesis. The metabolic markers for lipogenesis and metabolic rate could explain the high-fat concentration in several parts of the body composition of fish from Gilgel Gibe. Our results show that nutrition-related blood metabolite ratios are useful to understand the underlying metabolic events leading to the habitat-dependent differences in the growth of Nile tilapia, and by extension, other species., (© 2021. The Author(s).)

Functional fertilisers for hydroponics are in great demand. Herein, we isolated peptides from mackerel by-products, a valuable source of bioactive peptides. The pellet-phase fraction obtained after cold-acetone extraction exhibited plant growth-promoting activity in wheat hydroponics, and the presumed peptides were determined to be ≤ 1 kDa based on molecular weight cut-off and tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry analysis were employed for peptide purification and identification. Finally, two peptides were identified, both with linear structures, consisting of amino acid sequences TCGGQGR and KEAGAFIDR. At 1 mg/mL, the heptapeptide performed better than the nonapeptide in terms of wheat growth and health, but neither peptide exhibited antimicrobial activity. Only the heptapeptide displayed significant antioxidant activity, and this activity bioaccumulated in wheat leaves after 7 days of hydroponic growth. The heptapeptide did not match any known metabolites in PepBank, BIOPEP, UniProt or METLIN databases, and is therefore a novel peptide with potential as a functional fertiliser component., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The growth of fishes and their metabolism is highly variable in fish species and is an indicator for fish fitness. Therefore, somatic growth, as a main biological process, is ecologically and economically significant. The growth differences of two closely related salmonids, rainbow trout (Oncorhynchus mykiss) and maraena whitefsh (Coregonus maraena), have not been adequately studied as a comparative study and are therefore insufficiently understood. For this reason, our aim was to examine muscle growth in more detail and provide a first complex insight into the growth and muscle metabolism of these two fish species at slaughter size. In addition to skeletal muscle composition (including nuclear counting and staining of stem and progenitor cells), biochemical characteristics, and enzyme activity (creatine kinase, lactate dehydrogenase, isocitrate dehydrogenase) of rainbow trout and maraena whitefish were determined. Our results indicate that red muscle contains cells with a smaller diameter compared to white muscle and those fibres had more stem and progenitor cells as a proportion of total nuclei. Interestingly, numerous interspecies differences were identified; in rainbow trout muscle RNA content, intermediate fibres and fibre diameter and in whitefish red muscle cross-sectional area, creatine kinase activity were higher compared to the other species at slaughter weight. The proportional reduction in red muscle area, accompanied by an increase in DNA content and a lower activity of creatine kinase, exhibited a higher degree of hypertrophic growth in rainbow trout compared to maraena whitefish, which makes this species particularly successful as an aquaculture species., Competing Interests: The authors have declared that no competing interests exist.

Fish discards and by-products can be transformed into high value-added products such as fish protein hydrolysates (FPH) containing bioactive peptides. Protein hydrolysates were prepared from different parts (whole fish, skin and head) of several discarded species of the North-West Spain fishing fleet using Alcalase. All hydrolysates had moisture and ash contents lower than 10% and 15%, respectively. The fat content of FPH varied between 1.5% and 9.4% and had high protein content (69.8-76.6%). The amino acids profiles of FPH are quite similar and the most abundant amino acids were glutamic and aspartic acids. All FPH exhibited antioxidant activity and those obtained from Atlantic horse mackerel heads presented the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and Cu 2+ chelating activity. On the other hand, hydrolysates from gurnard heads showed the highest ABTS radical scavenging activity and Fe 2+ chelating activity. In what concerns the α-amylase inhibitory activity, the IC 50 values recorded for FPH ranged between 5.70 and 84.37 mg/mL for blue whiting heads and whole Atlantic horse mackerel, respectively. α-Glucosidase inhibitory activity of FPH was relatively low but all FPH had high Angiotensin Converting Enzyme (ACE) inhibitory activity. Considering the biological activities, these FPH are potential natural additives for functional foods or nutraceuticals.

This study was done to evaluate the effects of Bisphenol A (BPA) on Siberian sturgeon (Acipenser baerii). As liver is the main organ in the homeostatic adjustments to stress, we used a proteomics method to address molecular response in this tissue. Also, we compared the levels of vitellogenin in plasma and mucus to propose that the last one be a non-invasive method to analyze this biomarker. The fish received 1, 10, and 100 μg g -1  week -1 BPA intraperitoneally for two weeks. The samples were taken on days 0, 7, and 14. Plasma vitellogenin level increased as the highest value was recorded in the group with 100 μg g -1  week -1 of BPA. Changes in the mucus and blood vitellogenin showed a similar pattern, suggesting that mucus could be used for evaluating the changes in blood vitellogenin. Comparative proteomics was used to determine the proteome of the liver of A. baerii in the highest dose of BPA in comparison with the control. Sixteen proteins were identified that their expression changed at least twice between the studied groups. The proteomic results showed that BPA increased the expression of proteins involved in the detoxification and metabolism, activated glycolysis, and produced necrosis in the liver., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Declines across global fishery stocks forced aquaculture feed manufacturers to search for new and sustainable components. Therefore, the aim of study was assessing nutritional value and sensory properties of meat of common carp (Cyprinus carpio L.) fed for 116 days with two blends. The control feed contained 5% of fishmeal and vegetable oils (rapeseed and soybean) as sole fat sources. While in the experimental diet half of the fishmeal was replaced with a blend of microalgae (Spirulina sp., Chlorella sp.), macroalgae (Laminaria digitata) and vegetable oil was replaced with salmon oil. Proximate composition, energy value, fatty acid profile of meat, nutritional characteristics of fat and protein as well as culinary properties of fillets were assessed. Fillets of carp fed experimental diet had a higher level of protein, lower level of fat and energy value. Intramuscular fat of fish fed with the experimental diet had a better parameters of quality. Protein in the meat of fish from both groups was characterized by a high quality comparing to the protein standard. Our study showed that meat of carp fed with experimental feed enriched with sustainable and natural feed ingredients can be a sensorily attractive source of nutritious ingredients in the human diet., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In order to explore the common and unique physiological changes in tissues of juvenile barramundi Lates calcarifer in acidified water environment, RNA sequence analysis was used to analyze the molecular responses of liver, head kidney, and gill of juvenile barramundi in pH 7.4 and pH 8.1 seawater environment. The number of differential expression genes identified in liver, head kidney and gill were 860, 388 and 1792, respectively. Through functional enrichment analysis, the differential expression genes common to the three tissues were all related to immunity. Among the unique differential genes in the liver, pathways related to digestion, endocrine, and metabolism were enriched. Among the unique differential expression genes in gill, pathways related to genetic information processing, immunity and metabolism were enriched. The findings of the present study uncover the transcriptional changes in fish correspond to environmental pH change, and provide a better understanding on the biological process at molecular level to environmental pH adapting. This work highlights that assessments for the potential of estuarine fishes to cope with environmental pH change to develop the future conservation strategies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Consumer protection against food adulteration and misleading labelling is integrated into EU legislation, but accurate analysis of the meat content of farmed freshwater fish products is not possible because of the lack of established nitrogen factors for farmed common carp. The aim of this study was to determine nitrogen factors for farmed common carp Cyprinus carpio. Seven-hundred samples collected in 2018-2019 in three harvest seasons (March/April, Jun/July, and October/November) at seven locations in the Czech Republic were analysed for nitrogen, dry matter, protein, ash, and fat content according to standard ISO methods. The recommended nitrogen factor for fat-free common carp fillet with skin is 3.04 ± 0.13 and, for fillet without skin, 2.95 ± 0.12. Availability of nitrogen factors for common carp can help ensure that consumers are purchasing correctly labelled products.

Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides. The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We have developed a novel approach that involves inception-resnet network (IRN) modeling based on infrared spectroscopy (IR) for rapid and specific detection of the fish allergen parvalbumin. SDS-PAGE and ELISA were used to validate the new method. Through training and learning with parvalbumin IR spectra from 16 fish species, IRN, support vector machine (SVM), and random forest (RF) models were successfully established and compared. The IRN model extracted highly representative features from the IR spectra, leading to high accuracy in recognizing parvalbumin (up to 97.3%) in a variety of seafood matrices. The proposed infrared spectroscopic IRN (IR-IRN) method was rapid (~20 min, cf. ELISA ~4 h) and required minimal expert knowledge for application. Thus, it could be extended for large-scale field screening and identification of parvalbumin or other potential allergens in complex food matrices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Farlowella is the second richest genus in Loricariinae, broadly distributed in freshwater streams and rivers of South America. In this article, we aimed to expand on the cytogenetic and molecular data available for two allopatric populations of Farlowella hahni . Both populations had diploid chromosome number 58, but with karyotype differences, indicative of chromosomal rearrangements. C-banding showed large heterochromatic blocks at telomeric regions in acrocentric chromosomes in both populations. Fluorescence in situ hybridization (FISH) revealed a single 18S rDNA site in both populations and a single 5S rDNA site for individuals from lower Paraná River basin (native region) and multiple 5S rDNA sites for individuals from upper Paraná River basin (non-native region). Mitochondrial sequence analyses did not separate the two F. hahni populations. The cytogenetic and molecular data obtained are relevant in a preliminary study and suggested the existence of cryptic diversity and the hypothesis that at least two Farlowella lineages may coexist in the Paraná basin.

Phenotypic sex of an organism is determined by molecular changes in the gonads, so-called molecular sex differentiation, which should precede the rise of cellular or anatomical sex-distinguishing features. This study characterized molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with established XX/XY genotypic sex determination. Next generation sequencing was conducted on sablefish ovarian and testicular mRNAs to obtain sequences for transcripts associated with vertebrate sex determination and differentiation and early reproductive development. Gene-specific PCRs were developed to determine the distribution and ontogenetic gonadal expression of transcription, growth, steroidogenic and germline factors, as well as gonadotropin and steroid receptors. Molecular changes associated with sex differentiation were first apparent in both XY- and XX-genotype sablefish at ~ 60 mm in body length and prior to histological signs of sex differentiation. The earliest and most robust markers of testicular differentiation were gsdf, amh, dmrt1, cyp11b, star, sox9a, and fshr. Markedly elevated mRNA levels of several steroidogenesis-related genes and ar2 in differentiating testes suggested that androgens play a role in sablefish testicular differentiation. The earliest markers of ovarian differentiation were cyp19a1a, lhcgr, foxl2, nr0b1, and igf3. Other transcripts such as figla, zp3, and pou5f3 were expressed predominantly in XX-genotype fish and significantly increased with the first appearance and subsequent development of primary oocytes. This study provides valuable insight to the developmental sequence of events associated with gonadal sex differentiation in marine teleosts with XX/XY sex determination. It also implicates particular genes in processes of male and female development and establishes robust molecular markers for phenotypic sex in sablefish, useful for ongoing work related to sex control and reproductive sterilization., (Published by Elsevier B.V.)

Antifreeze proteins inhibit ice growth and are crucial for the survival of supercooled fish living in icy seawater. Of the four antifreeze protein types found in fishes, the globular type III from eelpouts is the one restricted to a single infraorder (Zoarcales), which is the only clade know to have antifreeze protein-producing species at both poles. Our analysis of over 60 unique antifreeze protein gene sequences from several Zoarcales species indicates this gene family arose around 18 Ma ago, in the Northern Hemisphere, supporting recent data suggesting that the Arctic Seas were ice-laden earlier than originally thought. The Antarctic was subject to widespread glaciation over 30 Ma and the Notothenioid fishes that produce an unrelated antifreeze glycoprotein extensively exploited the adjoining seas. We show that species from one Zoarcales family only encroached on this niche in the last few Ma, entering an environment already dominated by ice-resistant fishes, long after the onset of glaciation. As eelpouts are one of the dominant benthic fish groups of the deep ocean, they likely migrated from the north to Antarctica via the cold depths, losing all but the fully active isoform gene along the way. In contrast, northern species have retained both the fully active (QAE) and partially active (SP) isoforms for at least 15 Ma, which suggests that the combination of isoforms is functionally advantageous., Competing Interests: The authors have declared that no competing interests exist.

Interferon (IFN)γ is a pivotal cytokine that promotes and orchestrates innate cellular and adaptive cell-mediated immunity against intracellular pathogens. The capacity of T cells in mammals to produce IFNγ has been measured using specific antibodies in order to analyze cell-mediated immune responses against infection or immuno-stimulants. In fish, however, measurement of IFNγ protein levels has not been possible due to a lack of research tools. In the present study, therefore, we established antibodies that react with endogenous amberjack IFNγ. An enzyme-linked immunosorbent assay (ELISA) for IFNγ in amberjack species was developed using these antibodies. The ELISA could detect endogenous IFNγ at concentrations less than 100 pg/mL in PMA/ionomycin-stimulated leukocytes culture supernatant. IFNγ production was enhanced and lasted a long time following intracellular bacterial infection with Nocardia seriolae, which is thought to be targeted by cell-mediated immunity. These results demonstrate that quantification of IFNγ using the reported ELISA can be used to estimate the status of cell-mediated immunity in amberjack species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted and purified from the cartilages of skate and sturgeon. Their typical structure and physicochemical properties were evaluated by circular dichroism (CD), X-ray diffraction (XRD), and so on. Results showed that the extracted collagen was likely identified as collagen-II composed of three α-chains (135 kDa), with the typical peptide sequence of Gly-X-Y. It showed the collagen retained the native and intact triple helical structure, and its intensity ratio of the positive and negative absorption peaks (Rpn) was 0.19-0.25. In addition, the extracted collagen exhibited obvious self-assembly behavior with the concentration above 0.3 mg/mL, the adjustment of pH 7.4-7.6 and the NaCl concentration of 120 mmol/L. The critical aggregate mass concentrations of pepsin-soluble collagens from skate and sturgeon were 0.93 and 0.86 g/L, respectively. Therefore, collagens from skate and sturgeon cartilages have potential commercial application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The seasonal variation in chemical and lipid composition of chub mackerel (Scomber colias) was evaluated monthly over one year (proximate chemical composition and lipid profile: fatty acid (FA) and lipid classes distribution). Significant seasonal changes regarding fat content were noticed (1.3-10.3 g/100 g), with the lowest fat content obtained in February (during spawning period), and the highest in September. Regarding the FA profile, the main fluctuations were recorded in saturated (SFA) and polyunsaturated fatty acids (PUFA). The highest SFA content was registered between March and August (25.3-32.3%). PUFA (the most abundant group) reached its maximum percentual content between December and February (60.9 and 66.9%, respectively). In absolute terms, PUFA attained 5352.6 mg/100 g edible part in September, where 2473.8 mg/100 g of docosahexaenoic acid (DHA, C22:6n-3), representing 46.2% of total PUFA. DHA lowest level was 519.8 mg/100 g, registered in low-fat chub mackerel. Together DHA and EPA (eicosapentaenoic acid, C20:5n-3) represented 75% of the total PUFA and 84% of n-3 PUFA. Triacylglycerols (TAG) with 82.2-92.1% of total lipid content) and phospholipids (4.4-8.4%) were the main lipid classes. Polar lipid fraction (phospholipids), was predominantly constituted by PUFA (68.6-74.5%), mainly DHA (45.2-55.1%), with the highest percentage recorded in low-fat chub mackerel. High relative contents of PUFA (36.6-49.1%) were also found in TAG. Having into account the data obtained, chub mackerel is a privileged source of DHA even in a lean species whereby its consumption should be recommended as part a healthy dietary regime., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Atlantic salmon is often adulterated or substituted by rainbow trout with much lower price and quality. However, it is extremely difficult to distinguish Atlantic salmon and rainbow trout due to their similar appearance and close relationship in species. In the present work, untargeted and targeted proteomics approaches were both implemented to identify species-specific peptide biomarkers of Atlantic salmon and rainbow trout. Potential peptide biomarkers were obtained through matching HRMS data with UniProt database, screened by BLAST and then verified with real samples. Five peptide biomarkers were identified each for Atlantic salmon and rainbow trout. MRM method was established for quantitative measurement of rainbow trout Adulteration in Atlantic salmon, showing high sensitivity and repeatability. The biomarker peptide GDPGPGGPQGEQGVVGPAGISGDK was used for quantification. The limit of the detection (LOD) of adulteration of rainbow trout is 0.19%, and the limit of quantitation (LOQ) is 0.62%. Furthermore, this method was successfully applied to analyze a number of Atlantic salmon and Rainbow trout samples from different regions and different batches, as well as commercially available processed products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

This study was designed to investigate proteome changes in Japanese puffer fish (Takifugu rubripes) during short- and long-term frozen storage. In total, 1484 proteins were quantified, and 164 proteins were identified as differential abundance proteins (DAPs) in Japanese puffer fish from two frozen storage treatment groups (14 days and 60 days) compared with the fresh control group. Correlation analysis between the DAPs and quality traits of the puffer fish muscle showed that 106 proteins were correlated closely with colour and texture (hardness, elasticity, and chewiness). Bioinformatics analysis revealed and Western blot analysis verified that Putative prothymosin alpha species, Bridging integrator 3, NADH: the ubiquinone oxidoreductase subunit and Mx species are candidate biomarkers for puffer fish properties. This study offers valuable evidence to improve the quality control and monitoring of Japanese puffer fish during transportation and storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Three-dimensional printing (3DP) has attracted significant attention for its use in additive manufacturing techniques because it provides customizability and flexibility for fabricating structures with arbitrary shapes. Certain applications in the food and medicine industries require 3D printable materials that are both biocompatible and biodegradable. Consequently, this study reports 3D printable materials constructed from food-grade high internal phase emulsions (HIPEs). The studied HIPEs (phase ratio 85%) were stabilized by the efficient adsorption behavior of cod proteins (concentration range, 10-50 mg mL -1 ) at the oil-water interface. The stability of the oil-in-water HIPEs was improved by the formation of a concentration-dependent percentage of adsorbed proteins and cross-linking networks, and homogeneous and self-supporting structures were generated after 7 days of storage at 4 °C. The gel-like shear thinning rheological behavior induced by the cross-linking networks in the studied HIPEs can be tuned to obtain the desired printability and extrudability during 3DP. In the present study, the HIPEs stabilized with 50 mg mL -1 of cod proteins exhibited the highest printing resolution, gel strength, hardness, adhesiveness, and chewiness during 3DP. These food-grade HIPE inks have the potential to diversify the applications of 3DP in foods, cosmetics, drug delivery systems, and packaging materials.

An international project developed, quality-tested, and measured isotope-delta values of 10 new food matrix reference materials (RMs) for hydrogen, carbon, nitrogen, oxygen, and sulfur stable isotope-ratio measurements to support food authenticity testing and food provenance verification. These new RMs, USGS82 to USGS91, will enable users to normalize measurements of samples to isotope-delta scales. The RMs include (i) two honeys from Canada and tropical Vietnam, (ii) two flours from C3 (rice) and C4 (millet) plants, (iii) four vegetable oils from C3 (olive, peanut) and C4 (corn) plants, and (iv) two collagen powders from marine fish and terrestrial mammal origins. An errors-in-variables regression model included the uncertainty associated with the measured and assigned values of the RMs, and it was applied centrally to normalize results and obtain consensus values and measurement uncertainties. Utilization of these new RMs should facilitate mutual compatibility of stable isotope data if accepted normalization procedures are applied and documented.

Background: Fish is a major food and allergen source, requiring safety declarations on packages. Enzyme-linked immunosorbent assays (ELISAs) are often used to ensure that the product meets the required standards with regard to the presence of allergens. Over 1000 different fish species are traded and consumed worldwide, and they are increasingly provided by aquaculture. Up to 3% of the general population is at risk of sometimes fatal allergic reactions to fish, requiring strict avoidance of this commodity. The aim of this study is to evaluate the capacity of three commercially available ELISA tests to detect a wide variety of bony and cartilaginous fish and their products, which is essential to ensure reliable and safe food labeling., Results: The detection rates for 57 bony fish ranged from 26% to 61%. Common European and North American species, including carp, cod, and salmon species, demonstrated a higher detection rate than those from the Asia-Pacific region, including pangasius and several mackerel and tuna species. Among the 17 canned bony fish products, only 65% to 86% were detected, with tuna showing the lowest rate. None of the cartilaginous fish (n = 9), other vertebrates (n = 8), or shellfish (n = 5) were detected., Conclusions: We demonstrated that three commercial fish ELISA kits had a limited capacity to detect fish and their products. The complexity of fish as a protein source that is increasingly utilized means that there is an urgent need for improved detection methods. This is crucial for the food industry to provide safe seafood products and comply with international legislation. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

This project aimed to evaluate the effects of gallic acid (GA) and protocatechuic acid (PA) grafted onto chitosan (CS) on the improved quality of sea bass (Lateolabrax japonicus) during refrigerated storage. The incorporation of GA and PA onto CS (CS-g-GA and CS-g-PA) were achieved by the carbodiimide-mediated grafting procedure. Samples were treated with different solutions (deionized water [CK], 1% CS [m/v], 1% CS-g-GA [m/v], and 1% CS-g-PA [m/v]) for 10 min, which were then stored at 4 °C. Microbiological quality, including total viable counts (TVC), psychrophilic bacterial counts (PBC), Pseudomonas bacterial counts, and H 2 S-producing bacterial counts were measured. Physicochemical parameters, including pH, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA) value, water holding capacity (WHC), and K value, were measured. The changes in protein characteristics, including sulfhydryl groups (SH), Ca 2+ -ATPase activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and tertiary structure of protein were analyzed periodically, along with texture profile analysis (TPA). The results demonstrated that the CS copolymers treatment exhibited better preservation effects. The CS-g-GA and CS-g-PA treatments could significantly inhibit the growth of microorganisms and retard the increase of pH, TVB-N, TBA, WHC, and K-value during refrigerated storage compared with the CK and CS groups. Additionally, the CS-g-GA and CS-g-PA treatments could delay the protein oxidation by keeping a higher SH level and Ca 2+ -ATPase activity. The CS copolymers treatment could also extend the shelf life for another 6 days compared with that of CK. As a result, CS copolymers can be employed in a promising method for the preservation of sea bass. PRACTICAL APPLICATION: The incorporation of gallic acid and protocatechuic acid onto chitosan (CS-g-GA and CS-g-PA) showed superior antioxidant and antimicrobial activities when applied on sea bass. The CS-g-GA and CS-g-PA coatings could maintain the quality and freshness of refrigerated sea bass. Additionally, this research could provide a theoretical basis for the application of graft copolymers on the preservation of aquatic products., (© 2020 Institute of Food Technologists®.)

Consisting of 25 to 30% of protein in carp, water-soluble sarcoplasmic proteins lost in wash water, have been recovered and freeze-dried into a protein-rich powder. Study objectives were to evaluate protein quality and safety of a silver carp sarcoplasm derived protein powder (CSP) compared to commercial protein supplements, casein, and whey. In vivo protein quality assessment of CSP showed a lower (P < 0.05) protein digestibility corrected amino acid score compared to the commercial protein sources. Despite greater (P < 0.05) fecal amino acid excretion in casein-fed rats, there were no significant differences in liver and muscle amino acid profiles. All low (10% kcal) protein diets supported growth with the normal range. However, whey protein supplementation resulted in greater (P < 0.05) adiposity. CSP, casein, or whey-fed rats showed no differences in major organ weights, renal damage biomarkers, or bone indices. Collectively, results indicated CSP was safe with protein quality comparable to casein. PRACTICAL APPLICATION: As much as 40 percent of protein in fish can be lost due to sarcoplasmic protein solubilization in processing wash water. Silver carp sarcoplasm protein powder may have similar commercial potential as a sustainable and nutritious alternative to whey and casein proteins. This project aimed to verify the protein quality and safety of this economical protein source., (© 2020 Institute of Food Technologists®.)

Enkephalins are the pentapeptides involved in pain relief and neuroendocrine responses with high affinity for delta opioid receptors in vertebrates. In the present investigation, we studied the distribution of leucine-enkephalin-immunoreactive (L-ENK-ir) neurons in the brain of the cichlid fish Oreochromis mossambicus. Application of the antisera against L-ENK revealed the presence of numerous L-ENK-ir perikarya and fibres in subdivisions of the dorsal and the ventral telencephalon, the medial olfactory tract and the nucleus entopeduncularis, whereas intensely labelled L-ENK-ir fibres were noticed in the olfactory bulb. Furthermore, the presence of L-ENK-ir cells and dense accumulations of fibres in the preoptic area and its subdivisions, the nucleus preopticus pars magnocellularis and the nucleus preopticus pars parvocellularis suggested a role for this peptide in regulation of reproduction. While intensely labelled cells and fibres were found in the nucleus lateralis tuberis pars lateralis as well as the nucleus lateralis tuberis pars medialis, some L-ENK-ir fibres were seen at the hypothalamo-hypophyseal tract indicating the possible hypophysiotrophic role for this peptide. Numerous L-ENK-ir cells and dense network of fibres were observed in the subdivisions of the nucleus recess lateralis and the pretectal area, whereas intensely labelled thick network of L-ENK- fibres were found in the ventromedial thalamic nucleus, the sub-layers of the optic tectum and the rostral spinal cord. The widespread distribution of L-ENK-immunoreactivity in the olfactory bulb, the telencephalon, the diencephalon and the mesencephalon regions of the brain as well as the spinal cord suggests the possible involvement of this peptide in the regulation of diverse functions such as neuroendocrine, antinociceptive, visual and olfactory responses in O. mossambicus., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The objective of this study was to simultaneously obtain protein isolates and lipids from the dried powder of large yellow croaker (Pseudosciaena crocea) roes (pcRs) to achieve high-value utilization. Protein isolates and lipids were extracted simultaneously from pcRs by saline and acidic solutions. The purity of the protein isolates from the pcRs (pcRPIs) was greater than 70%, with vitellogenin, vitellogenin B and vitellogenin C as the main proteins. The lipids from pcRs (pcRLs) were mainly composed of triglycerides with high levels of EPA and DHA. The pcRPIs exhibited a higher surface hydrophobicity, water/oil holding capacity and emulsifying ability than those of the pcRs. Moreover, pcRPIs had a better oil holding capacity and emulsifying ability than soy protein isolate. These results suggest that protein isolates and lipids can be simultaneously extracted by saline and acidic solutions, and pcRPIs and pcRLs can be used as functional materials in the food industry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish.

The epidermal mucus protects fish against harmful environmental factors and the loss of physiological metabolites and water. It is an efficient barrier between the fish and the biosphere. The integrity of the skin mucus is thus of vital importance for the welfare and survival of the fish. Since excreted proteins and small molecules in the mucus can mirror the health status of the fish, it is a valuable matrix for monitoring stress, pathogen exposure, and nutritional effects. Several methods for sampling epidermal mucus from different fish species have previously been described, but information about their efficiency or the comparability of mucus analyses is lacking. In the present study, skin mucus from farmed Atlantic salmon was therefore sampled by three methods, including absorption or wiping with tissue paper, and scraping with a blunt blade, and the mucus proteome was analyzed by ultra-high pressure liquid chromatography high-resolution mass spectrometry. The measured protein contents, numbers, compositions and the observed data quality were compared between sampling methods. Furthermore, functional annotation and classification of the identified proteins was performed. The results showed that the three skin mucus sample types differed qualitatively as well as quantitatively. The absorbed mucus was the least tainted by proteins resulting from damage inflicted to the fish epidermis by the sampling procedure. Wiped mucus showed a better protein yield than absorbed and delivered a larger proteome of identifiable proteins, with less contamination from epithelial proteins than observed for scraped mucus. We recommend that future research of mucus should use the absorption method in cases, where it is important that the mucus is devoid of proteins from the underlying epithelium, and the wiping method, when protein yield is crucial or when the proteome of the outer epithelium is of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Bioprocessing of plant feedstuff can be a novel approach for reducing the overwhelming dependence on fish meal in aquaculture. The objective of this study was to evaluate the performance of Pacific white shrimp Litopenaeus vannamei fed solid-state fermented protein concentrates in order to replace fish meal in the diet., Results: In the first trial, a group of 15 shrimp (average 3.88 g) were randomly distributed into aquaria in triplicate according to the experimental diets. Ten isonitrogenous (400 g kg -1 CP) and isolipidic (90 g kg -1 CL) diets were formulated to contain high-protein fish meal (HFM) and low-protein fish meal (LFM), and four types of bioprocessed protein concentrates (BPCs) as a replacement of fish meal (BPC-A, -B, -C and -D) each at 30% and 50% FM replacement levels. BPC-A was a solid-state fermented mixture of soybean and corn gluten meals; BPC-B was pre-treated acid-hydrolyzed BPC-A; BPC-C and BPC-D were BPC-A + 2% shrimp soluble extract (SSE) and BPC-B + 2% SSE, respectively. After 8 weeks, shrimp fed the HFM, BPC-B, BPC-C and BPC-D diets showed significantly higher growth performance at 30% FM replacement than those of shrimp fed the BPC diets at 50% FM replacement. Interestingly, shrimp fed the BPC-D diet could replace up to 50% FM replacement. In the second trial, the results show that apparent digestibility coefficients of feeds and apparent digestibility coefficients of ingredients for crude protein were significantly higher in fish fed the BPC-B, BPC-C and BPC-D diets., Conclusions: The results demonstrated successful partial replacement of high-protein fish meal using high-quality fermented protein concentrates from plant sources. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection., Competing Interests: The authors have declared that no competing interests exist.

Disulfide bonds are fundamental in establishing Ig structure and maintaining Ig biological function. Here, we analysed disulfide bonds and free cysteine in three grass carp IgM isoforms (monomeric, dimeric/trimeric, and tetrameric IgM) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results revealed that Cys 574 residue status at the C-terminal tail differed substantially in monomeric IgM in comparison with polymeric IgM, Cys 574 was found as free thiol in monomeric IgM, while it formed disulfide linkages in dimeric/trimeric and tetrameric IgM. Five intra-chain disulfide bonds in the CH1~CH4 and CL1 domains, as well as one H-H and one H-L inter-chain disulfide linkages, were also observed and shown identical connectivity in monomeric, dimeric/trimeric, and tetrameric IgM. These findings represent the first experimental assignments of disulfide linkages of grass carp IgM and reveal that grass carp IgM isoform formation is due to alternative disulfide bonds connecting the Cys 574 residue at the C-terminal tail., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The TLRs of teleost fishes have distinct features and are highly diverse, but the duplication characteristics and expression patterns of the tlr22 gene remain unclear. Here, we identified paralogous tlr22 genes in 13 teleost fishes by screening available fish genomic resources and using molecular cloning. We then conducted comprehensive bioinformatics analyses and investigated spatiotemporal differences in the expression patterns of the tlr22 genes in G. eckloni. The results indicated that more than three paralogous tlr22 genes were possessed by some teleost fishes. Of these, tlr22c is specific to some subfamilies of the Cyprinidae (e.g., Barbinae, Cyprininae, Schizothoracinae, and Leuciscinae). Phylogenetic and syntenic analyses showed that the paralogous tlr22 genes originated from two single-gene duplication events. Molecular clock calculations dated the two gene duplication events at 49.5 and 39.3 MYA, which is before the common carp-specific genome duplication event and well after the fish-specific genome duplication. Gene duplication of tlr22 was followed by gene loss or pseudogene events in certain lineages. Spatiotemporal expression differences between the three duplicated tlr22 genes from G. eckloni suggested that these genes diverged functionally after gene duplication., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Omics technologies have been widely applied in different fields, among which, proteomics is gaining increasing interest for its application to the authenticity of food products. MS, typically coupled with LC, represents a key technique for proteomics-related studies dedicated to fish and other seafood products by using a bottom-up approach. Objective and Methods: In this paper, the optimization of an untargeted proteomics-based method using LC separation and MS detection relying on a quadrupole time-of-flight mass spectrometer is described and applied to the analysis of Canadian farmed and wild-type salmon, followed by statistical analysis based on principal component (PC) analysis. Results and Conclusions: This untargeted approach, using a data-independent acquisition MS scheme, demonstrated the ability to effectively discriminate salmon belonging to the two classes. Furthermore, selected peptides showing high loadings on PC1 could represent potential candidate peptide markers able to discriminate farmed from wild-type salmon samples in the future.

Background: Fish and fish products are one of the most important food sources of high commercial interest. The global food trade and the associated risks are constantly presenting new challenges to consumer protection and public authorities, which, among other things, demand state-of-the-art analytical methods to ensure food authenticity. Objective: The establishment of MS-based strategies plays a decisive role alongside the (further) development of ELISA- or DNA-oriented methods. Methods: In the present work, therefore, the development and in-house validation of an LC-MS and LC-MS/MS-based assay for authenticity testing of certain fish species is described. Results: Based on the execution of a validated bottom-up LC-electrospray-MS and MS/MS assay and multivariate analysis, the commercially available species Lutjanus malabaricus (red snapper) and Sebastes spp. (redfish) are distinguished from each other, whereas an additional 68 samples [nine additional marine species such as pangasius ( Pangasianodon hypophthalmus ), salmon ( Salmo salar ), turbot ( Scophthalmus maximus ), plaice ( Pleuronectes platessa ), sole ( Solea solea ), lemon sole ( Glyptocephalus cynoglossus ), halibut ( Reinhardtius hypoglossoides ), red salmon ( Oncorhynchus nerka ), and great scallop ( Pecten jacobaeus )] served as blinded negative controls to ensure the specificity of the assay. Conclusions and Highlights: A promising LC-MS and LC-MSMS based assay has been developed that could enable the detection of fish fraud at the protein level in the future.

Rationale: Authentication of fish is of importance in the view of toxins, allergen warnings and economic fraud control. Traditional methods in the authentication of fish, e.g. morphological, genetic and proteomic analysis, are either at low throughput or at high-cost., Methods: A high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS)-based approach was developed to analyze biomaterials from fish skin, and mass spectra from different fish species were compared by chemometric methods to differentiate fish species., Results: A total of 51 fish samples were used to generate more than 150 fingerprinting mass spectra. The fish belonging to the same genus can be identified at species level. A mass spectral database of different fishes can be built as reference for authentication. The analysis can be performed based on micrograms of fish-skin sample and accomplished in 1-3 hours., Conclusions: The developed strategy holds potential to be applied to fish authentication in the fishing industry and as a scientific method to avoid mislabeling. It has promise to be practically used for fast and effective identification of closely related fish species to guarantee the quality of fishery products to consumers., (© 2019 John Wiley & Sons, Ltd.)

A liquid chromatography-tandem mass spectrometry method for the identification of marker peptides of aquatic product allergens and quantitative detection of multiple allergens in meat products and seasonings was developed. The samples were prepared by protein extraction, protein purification, and trypsin hydrolysis. The proteins and peptides were identified using ProteinPilot by the data analysis of the ion spectrum of polypeptide fragments using ultra-performance liquid chromatography-quadrupole/electrostatic orbitrap high-resolution mass spectrometry (UPLC-Q/Exactive-HRMS). The identification of 30 species-specific marker peptides in Penaeus vannamei, Eriocheir, Scylla serrata, Thunnus thynnus , and Atlantic salmon by comparison of the basic local alignment search tool (BLAST) with the UniProt database was achieved. The verification and multiple reaction monitoring (MRM) quantitative studies of these marker peptides were performed using a triple quadrupole mass spectrometry (UPLC-QqQ-MS) system. The proposed method showed a good linear relationship in the range of 5-250 mg/kg. The limits of quantitation and observed recoveries were in the range of 2-3.5 mg/kg and 88.7%-110.2%, respectively. This method presents various advantages such as good repeatability and high throughput, suitability for rapid screening, and quantitative analysis of seven aquatic allergens in meat products and seasonings.

Ghrelin (orexigenic) and nesfatin-1 (anorexigenic) are two peptides with opposing actions on food intake regulation and are mainly expressed in the hypothalamus and gut of mammals and fish. Both are involved in the regulation of a wide range of physiological processes in vertebrates, including metabolism, growth, and reproduction. However, the anatomical relationship between these peptides and the nutrient assimilation processes are not well understood. Thus, the aim of this work was to determine the localization of ghrelin, nesfatin-1, and several enzymes involved in the digestive process (lipoprotein lipase, aminopeptidase A, trypsin, and sucrase-isomaltase) in the intestine of pejerrey (Odontesthes bonariensis), a species with commercial importance in South America. We observed co-localization of ghrelin and nesfatin-1 in enteroendocrine cells, absorptive cells, and in cells of the lamina propia. Approximately half of the cells displaying ghrelin-like immunoreactivity co-localized the NUCB2/nesfatin-1-like signal. In addition, both peptides showed co-localization with lipoprotein lipase, aminopeptidase A, trypsin, or sucrase-isomaltase. All digestive enzymes except for aminopeptidase A and trypsin, showed high co-localization (68-88%) with both ghrelin-like and NUCB2/nesfatin-1-like signals in absorptive, enteroendocrine, and lamina propria cells. Together, our results provide immunohistochemical evidence supporting a role for both ghrelin and NUCB2/nesfatin-1 in the regulation of nutrient assimilation in fish. Anat Rec, 302:973-982, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Collagen was extracted from bigeye tuna ( Thunnus obesus ) skins by salting-out (PSC-SO) and isoelectric precipitation (PSC-IP) methods. The yield of the PSC-IP product was approximately 17.17% (dry weight), which was greater than the yield obtained from PSC-SO (14.14% dry weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that collagen from bigeye tuna skin belongs to collagen type I. Inductively coupled plasma mass spectrometry results indicate that the heavy metal abundance in PSC-IP was lower than the maximum acceptable amounts according to Chinese regulatory standards. In addition, results from a methylthiazolyldiphenyl-tetrazolium bromide assay and an in vitro scratch assay demonstrated that PSC-IP could promote the proliferation and migration of NIH-3T3 fibroblasts. Overall, results suggest PSC-IP could be used to rapidly extract collagen from marine by-products instead of traditional salting-out methods. Collagen from bigeye tuna skin may also have strong potential for cosmetic and biomedical applications.

Astyanax is a genus with a wide distribution ranging from the south United States to north of Patagonia (Argentina). The available cytogenetic data on Astyanax indicate a high karyotypic diversity, with diploid number of 36-52 chromosomes, presence of B chromosomes, heterochromatin polymorphism, and variations with respect to the number and localization of nucleolar organizer regions (NORs) and 18S and 5S ribosomal DNA sites. In the present study, we estimated the evolutionary history and times of divergence for 10 nominal Astyanax species from the South and Central/North American (Cna) continents, which present distinct chromosomal characteristics, based on molecular clocks inferred from mitochondrial DNA sequence. The molecular clock results indicate the origin of three distinct clades (Humeral dark spot [Hds]; Diffuse humeral spot [Dhs]; Cna group) during the late Miocene about 11.2 million years ago (Mya). Thus, Astyanax mexicanus (Cna) represent a species that diverged a long time ago (∼8.6 Mya) from the Hds group, and Astyanax schubarti is the oldest species (∼6.5 Mya) among the Dhs species.

Rhamdia quelen, a species of Heptapteridae, is considered to be a complex because of taxonomic and phylogenetic inconsistencies. Determining the physical location of repetitive DNA sequences on the chromosomes and the DNA barcode might increase our understanding of these inconsistencies within different groups of fish. To this end, we analyzed R. quelen populations from two river basins in Brazil, Paraguay and Parana, using DNA barcoding and different chromosomal markers, including U2 snDNA, which has never been analyzed for any Rhamdia species. Cytochrome c oxidase I gene sequence analysis revealed a significant differentiation among populations from the Miranda and Quexada rivers, with genetic distances compatible to those found among different species in neotropical fishes. Our results, in general, revealed a conservative chromosomal evolution in R. quelen and a differential distribution of some markers, such as 5S rDNA and U2 snDNA, in different populations. We suggest that R. quelen must undergo a major revision in its morphological, genetic, and cytogenetic molecular and taxonomic structure to elucidate possible operational taxonomic units.

The effects of low marine ingredient diets supplemented with graded levels (L1, L2, L3) of a micronutrient package (NP) on growth and metabolic responses were studied in diploid and triploid salmon parr. Diploids fed L2 showed significantly improved growth and reduced liver, hepatic steatosis, and viscerosomatic indices, while fish fed L3 showed suppressed growth rate 14 weeks post feeding. In contrast, dietary NP level had no effect on triploid performance. Whole body mineral composition, with exception of copper, did not differ between diet or ploidy. Whole fish total AAs and N-metabolites showed no variation by diet or ploidy. Free circulating AAs and white muscle N-metabolites were higher in triploids than diploids, while branch-chained amino acids were higher in diploids than triploids. Diploids had higher whole body α-tocopherol and hepatic vitamins K 1 and K 2 than triploids. Increased tissue B-vitamins for niacin and whole-body folate with dietary NP supplementation were observed in diploids but not triploids, while whole body riboflavin was higher in diploids than triploids. Hepatic transcriptome profiles showed that diploids fed diet L2 was more similar to that observed in triploids fed diet L3. In particular, sterol biosynthesis pathways were down-regulated, whereas cytochrome P450 metabolism was up-regulated. One‑carbon metabolism was also affected by increasing levels of supplementation in both ploidies. Collectively, results suggested that, for optimised growth and liver function, micronutrient levels be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when fed low marine ingredient diets. The study also suggested differences in nutritional requirements between ploidy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The shortage of fish oil (FO) leads to the extensive use of vegetable oil (VO) in marine fish diets. High replacement percentage of dietary FO by VO induced pro-inflammatory response of adipose tissue (AT) and liver tissue (LT) in large yellow croaker (Larimichthys crocea). Mammalian studies showed that the secretion of cytokines by AT affected the immune response of LT. To investigate whether or not the inflammation response of LT is related to AT in large yellow croaker, LT and AT cells from fish fed FO diet (FOL and FOA) and VO diet (VOL and VOA) were co-cultured in a trans-well system, which resulted in an assembly of the two cells types sharing the culture medium but being separated by the membrane of the insert. Co-culture of FOL and FOA was selected as the control group (FOL-FOA). Results indicated that, when compared with the control group, the expression of pro-inflammatory genes (toll like receptors [TLRs], tumour necrosis factor α [TNFα], interleukin 1β [IL1β], suppressor of cytokine signalling 3 [SOCS3] and cyclooxygenase 2 [COX2]) in FOL was significantly increased in the co-culture group of FOL and VOA (FOL-VOA), while the expression of anti-inflammatory genes (arginase I [ArgI] and transforming growth factor β1 [TGFβ1]) in FOL was significantly depressed. On the contrary, a significantly depressed expression of pro-inflammatory genes (TLRs, TNFα, IL1β and COX2) and increased expression of anti-inflammatory genes (interleukin 10 [IL10]) in VOL was observed in the co-culture group of VOL and FOA (VOL-FOA) when compared with the co-culture group of VOL and VOA (VOL-VOA). The change of immune-related gene expressions in LT cells was attributed to nuclear factor κB (NF-κB) signalling since the expression of the p65 protein was observed to show a similar trend to the expression of pro-inflammatory genes. It is speculated that dietary VO increased the secretion of cytokines, which induced pro-inflammatory response in LT cells. These ex vivo results indicate that AT plays a vital role in LT pro-inflammatory response in fish fed VO diet., (Copyright © 2018. Published by Elsevier Ltd.)