Lactic acid bacteria (LAB) isolated from freshwater fish (hatcheries and captures) from Paraná river (Argentina) were analyzed by using culture-dependent approaches. The species belonging to Carnobacterium (C.) divergens, C. inhibens, C. maltaromaticum, C. viridans and Vagococcus (V.) salmoninarum were identify as predominant by RAPD-PCR and 16 s rRNA gene sequencing. C. maltaromaticum (H-17, S-30, B-42 and S-44) grew in raw fish extract and slightly reduced the medium pH (5.81-5.91). These strains exhibited moderate fish sarcoplasmic protein degradation (≤ 73 %) releasing small peptides and free amino acids, being alanine, glycine, asparagine and arginine concentrations increased in a higher extent (17.84, 1.47, 1.26 and 0.47 mg/100 mL, respectively) by S-44 strain at 96 h incubation. Interestingly C. maltaromaticum H-17 was able to inhibit Listeria monocytogenes. Results suggest that these strains would contribute to the development of new safe and healthy fishery products with improved nutritional and sensory characteristics.

Because hemoglobins (Hbs) of all animal species have the same heme group, differences in their properties, including oxygen affinity, electrophoretic mobility, and pH sensitivity, must result from the interaction of the prosthetic group with specific amino acid residues in the primary structure. For this reason, fish globins have been the object of extensive studies in the past few years, not only for their structural characteristics but also because they offer the possibility to investigate the evolutionary history of Hbs in marine and freshwater species living in a large variety of environmental conditions. For such a purpose, phylogenetic analysis of globin sequences can be combined with knowledge of the phylogenetic relationships between species. In addition, Type I functional-divergence analysis is aimed toward predicting the amino acid residues that are more likely responsible for biochemical diversification of different Hb families. These residues, mapped on the three-dimensional Hb structure, can provide insights into functional and structural divergence.

TRIM proteins have recently emerged as novel players in antiviral defense. TRIM proteins contain a tri-partite motif, composed of a RING zinc finger, one or two B-boxes and a coiled-coil domain. Many members of this large protein family of E3 ubiquitin ligases catalyze the attachment of ubiquitin to a substrate protein, an activity dependent on the RING domain. We earlier made a full description of the TRIM gene family in zebrafish and pufferfish and identified three multigene TRIM subsets, a feature unique to fish. To determine their biological role, we further characterized members of the finTRIM subset. FinTRIM gene expression was studied during development and in multiple tissues in adult rainbow trout. Upregulation of a large number of finTRIM upon viral stimulation suggests they are involved in antiviral immunity. We also demonstrate that two finTRIM members display E3 ubiquitin ligase activity, indicating that finTRIMs could regulate antiviral signaling through ubiquitination.

Acute-phase protein (APPs) serum levels have been studied in many human diseases, and their components contribute to host defense during the evolution of infectious diseases by acting as part of the innate immune system. Based on the importance of establishing new experimental models, the present investigation evaluated the modulation of APPs following inflammatory stimulus by the inoculation of Aeromonas hydrophila in tilapias. Fish were sampled 6 and 24 hours post-infection. Tilapias presented increase of positive APPs such as ceruloplasmin, haptoglobin, alpha-2-macroglobulin and complement C3, as well as decrease of negative APPs such as albumin and transferrin. The protein response of tilapias during the course of bacterial infection showed correlation with the kinetics of cellular accumulation in the inflamed focus with significant increase of granulocytes, thrombocytes, lymphocytes and macrophages. However, granulocytes were the predominant cells, associated with increment in the reactive oxygen species (ROS) production. Showing responses similar to those observed in humans, the modulation of APPs and the kinetics of cellular accumulation in the exudate demonstrate the feasibility of this alternative experimental model for advances and studies to understand changes in pathophysiological mechanisms of acute inflammatory reaction due to bacterial infection.

Molecular compatibility between gametes is a prerequisite for successful fertilization. As long as a sperm and egg can recognize and bind each other via their surface proteins, gamete fusion may occur even between members of separate species, resulting in hybrids that can impact speciation. The egg membrane protein Bouncer confers species specificity to gamete interactions between medaka and zebrafish, preventing their cross-fertilization. Here, we leverage this specificity to uncover distinct amino acid residues and N-glycosylation patterns that differentially influence the function of medaka and zebrafish Bouncer and contribute to cross-species incompatibility. Curiously, in contrast to the specificity observed for medaka and zebrafish Bouncer, seahorse and fugu Bouncer are compatible with both zebrafish and medaka sperm, in line with the pervasive purifying selection that dominates Bouncer's evolution. The Bouncer-sperm interaction is therefore the product of seemingly opposing evolutionary forces that, for some species, restrict fertilization to closely related fish, and for others, allow broad gamete compatibility that enables hybridization.

Pentraxins are a family of pentameric serum proteins that have been conserved in evolution and share sequence homology, similar subunit assembly and the capacity for calcium-dependent ligand binding. The classical pentraxins are human C-reactive protein (CRP) and serum amyloid P component (SAP). The sequence homology and gene organization indicate that they arose from a gene duplication of an ancestral pentraxin gene. They are usually isolated based on their affinity for phosphorylcholine and agarose, respectively. We have used this method for isolation of pentraxin-like proteins from normal serum of Atlantic salmon (Salmo salar), common wolffish (Anarhichas lupus), cod (Gadus morhua) and halibut (Hippoglossus hippoglossus). Although pentraxin structures have not been verified, the isolated proteins all appear to be pentraxin-like based on their binding specificity, molecular weight of subunits, cross-reactivity with antibodies to human pentraxins and N-terminal amino acid sequences. However, with the described method only one pentraxin-like protein was detected in each of the fish species.

The evolution of antifreeze glycoproteins has enabled notothenioid fish to flourish in the freezing waters of the Southern Ocean. Whereas successful at the biodiversity level to life in the cold, paradoxically at the cellular level these stenothermal animals have problems producing, folding, and degrading proteins at their ambient temperatures of -1.86 °C. In this first multi-species transcriptome comparison of the amino acid composition of notothenioid proteins with temperate teleost proteins, we show that, unlike psychrophilic bacteria, Antarctic fish provide little evidence for the mass alteration of protein amino acid composition to enhance protein folding and reduce protein denaturation in the cold. The exception was the significant overrepresentation of positions where leucine in temperate fish proteins was replaced by methionine in the notothenioid orthologues. We hypothesize that these extra methionines have been preferentially assimilated into the genome to act as redox sensors in the highly oxygenated waters of the Southern Ocean. This redox hypothesis is supported by analyses of notothenioids showing enrichment of genes associated with responses to environmental stress, particularly reactive oxygen species. So overall, although notothenioid fish show cold-associated problems with protein homeostasis, they may have modified only a selected number of biochemical pathways to work efficiently below 0 °C. Even a slight warming of the Southern Ocean might disrupt the critical functions of this handful of key pathways with considerable impacts for the functioning of this ecosystem in the future.

Chemical signals are produced by aquatic organisms following predatory attacks or perturbations such as parasitic infection. Ectoparasites feeding on fish hosts are likely to cause release of similar alarm cues into the environment due to the stress, wounding, and immune response stimulated upon infection. Alarm cues are often released in the form of proteins, antimicrobial peptides, and immunoglobulins that provide important insights into bodily function and infection status. Here we outline a noninvasive method to identify potential chemical cues associated with infection in fish by extracting, purifying, and characterizing proteins from water samples from cultured fish. Gel free proteomic methods were deemed the most suitable for protein detection in saline water samples. It was confirmed that teleost proteins can be characterized from water and that variation in protein profiles could be detected between infected and uninfected individuals and fish and parasite only water samples. Our novel assay provides a noninvasive method for assessing the health condition of both wild and farmed aquatic organisms. Similar to environmental DNA monitoring methods, these proteomic techniques could provide an important tool in applied ecology and aquatic biology.

We partially characterized proteins that inhibit DNA acid precipitation from various fish eggs (Sparus aurata, Dicentrarchus labrax, Mugil cephalus and Zeus faber). The active proteins were purified by acetone fractionation. The activity was found to be heat resistant. Of bivalent cations tested only Co(2+) and Cu(2+) exerted a profound promoting effect in the activity from all fish. The protein fraction from Sparus aurata inhibited DNA synthesis in PCR performed by different DNA polymerases. The possible role of DNA protective proteins in fish egg physiology is discussed.

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28 °C to 18 °C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%-90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%-100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.

Type I interferon and apoptosis elicit multifaceted effects on host defense and various diseases, such as viral infections and cancers. However, the gene/protein network regulating type I interferon and apoptosis has not been elucidated completely. In this study, we selected grass carp (Ctenopharyngodon idella) as an experimental model to investigate the modulation of RNASEK on the secretion of type I interferon and apoptosis. We first cloned two paralogs RNASEK-a and -b in grass carp, defined three exons in each gene, and found the length of both coding regions is 306 bp with 73.27% of protein homology. The protein sequences of the two paralogs are highly conserved across species. Two proteins were mainly localized in early and late endosomes and endoplasmic reticulum. Further, quantitative real-time PCR demonstrated that dsRNA poly I:C and grass carp reovirus upregulated RNASEK-a and -b in grass carp cells and tissues. Overexpression of RNASEK-a and -b individually induced type I interferon expression and the phosphorylation of IRF3/IRF7 shown by Western blot and immunofluorescent staining, increased Bax/Bcl-2 mRNA ratio, DNA fragmentations, TUNEL-positive cells, and the proportion of Annexin V-positive signals in flow cytometry, and activated eIF2α, opposite to that observed when RNASEK-a and -b were knocked down in multiple cell types. Taken together, we claim for the first time that fish paralog proteins RNASEK-a and -b enhance type I interferon secretion and promote apoptosis, which may be involved in the phosphorylation of IRF3/IRF7 and eIF2α, respectively. Our study reveals a previously unrecognized role of RNASEK as a new positive regulator of type I interferon and apoptosis.

A high circulating cholesterol concentration is considered an important risk factor for the development of CVD. Since lean fish intake and fish protein supplementation have been associated with lower cholesterol concentration in some but not all clinical studies, the main aim of this study was to investigate the effect of diets containing proteins from fish muscles and fish by-products on the serum/plasma total cholesterol (TC) concentration in rodents. A systematic literature search was performed using the databases PubMed, Web of Science and Embase, structured around the population (rodents), intervention (type of fish and fraction, protein dose and duration), comparator (casein) and the primary outcome (circulating TC). Articles were assessed for risk of bias using the SYRCLE's tool. A meta-analysis was conducted in Review Manager v. 5·4·1 (the Cochrane Collaboration) to determine the effectiveness of proteins from fish on the circulating TC concentration. Thirty-nine articles were included in the systematic review and meta-analysis, with data from 935 rodents. The risk of bias is unclear since few of the entries in the SYRCLE's tool were addressed. Consumption of proteins from fish resulted in a significantly lower circulating TC concentration when compared with control groups (mean difference -0·24 mmol/l, 95 % CI - 0·34, -0·15, P < 0·00001), with high statistical heterogeneity (I2 = 71 %). To conclude, proteins from fish muscles and by-products show promise as a functional dietary ingredient or supplement by preventing high cholesterol concentration in rodents, thus reducing one of the most important risk factors for developing CVD.

The disposal of acid whey (Aw), a by-product from fermented products, is a problem for the dairy industry. The fishery industry faces a similar dilemma, disposing of nearly 50% of fish processed for human consumption. Economically feasible and science-based alternatives are needed to overcome this problem. One possible solution is to add value to the remaining nutrients from these by-products. This study focuses on the breakdown of nutrients in controlled fermentations of Aw, fish waste (F), molasses (M), and a lactic acid bacteria (LAB) strain (Lr). The aim was to assess the dynamic variations in microbial diversity and the biochemical changes that occur during fermentation. Four treatments were compared (AwF, AwFM, AwFLr, and AwFMLr), and the fermentation lasted 14 days at 22.5 °C. Samples were taken every other day. Colorimetric tests for peptide concentrations, pH, and microbial ecology by 16S-v4 rRNA amplicon using Illumina MiSeq were conducted. The results of the microbial ecology showed elevated levels of alpha and beta diversity in the samples at day zero. By day 2 of fermentation, pH dropped, and the availability of a different set of nutrients was reflected in the microbial diversity. The fermentation started to stabilize and was driven by the Firmicutes phylum, which dominated the microbial community by day 14. Moreover, there was a significant increase (3.6 times) in peptides when comparing day 0 with day 14, making this treatment practical and feasible for protein hydrolysis. This study valorizes two nutrient-dense by-products and provides an alternative to the current handling of these materials.

Diet is considered the most influential factor in modulating the gut microbiota but how dietary protein sources differ in their modulatory effects is not well understood. In this study, soy, meat (mixture of beef and pork), and fish proteins (experiment 1) and soy, milk (casein), and egg proteins (experiment 2) were fed to rats with cellulose (CEL) and raffinose (RAF); the microbiota composition and short-chain fatty acid concentration in the cecum were determined. Egg protein feeding decreased the concentration of acetic acid and the richness and diversity of the cecum microbiota. RAF feeding increased the concentrations of acetic and propionic acids and decreased the richness and diversity of the cecum microbiota. When fed with CEL, the abundance of Ruminococcaceae and Christensenellaceae, Akkermansiaceae and Tannerellaceae, and Erysipelotrichaceae enhanced with soy protein, meat and fish proteins, and egg protein, respectively. The effects of dietary proteins diminished with RAF feeding and the abundance of Bifidobacteriaceae, Erysipelotrichaceae, and Lachnospiraceae increased and that of Ruminococcaceae and Christensenellaceae decreased regardless of the protein source. These results indicate that, although the effect of prebiotics is more robust and distinctive, dietary protein sources may influence the composition and metabolic activities of the gut microbiota. The stimulatory effects of soy, meat, and egg proteins on Christensenellaceae, Akkermansiaceae, and Erysipelotrichaceae deserve further examination to better elucidate the dietary manipulation of the gut microbiota.

The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrP(C), into an aberrant and partially proteinase K resistant isoform, PrP(Sc). Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) sperm are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO sperm can bind to the oolemma, they show the fusion defect, resulting that Dcst1 KO males become almost sterile and Dcst2 KO males become sterile. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO sperm are motile and can approach the egg, but are defective in binding to the oolemma. Furthermore, we find that DCST1 and DCST2 interact with each other and are interdependent. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species, highlighting their crucial role as conserved factors in fertilization.

Population growth has led to increased global discharges of wastewater. Contaminants that are not fully removed during wastewater treatment, such as pharmaceuticals and personal care products (PPCPs), may negatively affect aquatic ecosystems. PPCPs can bioaccumulate causing adverse health effects and behavioural changes in exposed fish. To assess the impact of PPCPs on wild fish, and to assess whether caged fish could be used as a surrogate for resident wild fish in future monitoring, we caged goldfish in a marsh affected by discharges of wastewater effluents (Cootes Paradise, Lake Ontario, Canada). We collected plasma from resident wild goldfish, and from goldfish that we caged in the marsh for three weeks. We analyzed the plasma proteome and metabolome of both wild and caged fish. We also compared proteomic and metabolic responses in caged and wild fish from the marsh to fish caged at a reference site (Jordan Harbour Conservation Area). We identified significant changes in expression of over 250 molecules that were related to liver necrosis, accumulation and synthesis of lipids, synthesis of cyclic AMP, and the quantity of intracellular calcium in fish from the wastewater affected marsh. Our results suggest that PPCPs could be affecting the health of wild fish populations.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

Two-dimensional difference gel electrophoresis (2D DIGE) was applied to investigate the impact of slaughtering on the postmortem integrity of muscle tissue proteins in European sea bass (Dicentrarchus labrax). Three different slaughtering techniques were evaluated: asphyxia in air (AA), asphyxia in ice (AI), and spinal cord severance (SCS). Principal components analysis (PCA) revealed a significant divergence of SCS samples, whereas AA and AI samples, although grouped separately, were less divergent and could be included in a single asphyxia cluster. In terms of single proteins, the most significant impact was seen on nucleoside diphosphate kinase B, which was consistently less affected when fish were slaughtered by SCS as compared to asphyxia. Integrity of the sarcomeric proteins myosin heavy chain and myosin binding protein C and of the cytosolic proteins fructose biphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and enolase 1 was also better preserved upon SCS slaughtering. Most interestingly, the influence on muscle protein integrity could be detected since the early postmortem phase. In conclusion, slaughtering by SCS preserves protein integrity better than death by asphyxia, either in ice or in air. Both asphyxia conditions are comparably more adverse than SCS to muscle protein integrity, although a general trend favoring AI over AA is observed.

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (tn: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response.