2D-DIGE proteomic analysis of blood plasma reveals changes in immune- and stress-associated proteins following hormonal stimulation of carp males.

In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2 <-1.2-fold change, P < 0.05) in the abundance of 30 spots (14 up- and 16 downregulated) and 44 spots (28 up- and 16 downregulated) upon CPE and Ovopel treatment, respectively. The most significantly affected pathways were acute phase response signaling, the coagulation system, LXR/RXR and FXR/RXR activation; however, there were different sets of proteins in Ovopel- and CPE-treated males. The majority of differentially abundant proteins were involved in the regulation of the immune defense response, the response to stress, and complement activation. Moreover hormonal stimulation with CPE markedly increased the bactericidal activity of blood and both preparations caused profound changes in gene expression in hematopoietic organs. This work is important in understanding the biological processes behind the protein-based response to hormonal stimulation of sperm production in fish.

Health Surveillance of Wild Brown Trout (Salmo trutta fario) in the Czech Republic Revealed a Coexistence of Proliferative Kidney Disease and Piscine Orthoreovirus-3 Infection.

The population of brown trout (Salmo trutta fario) in continental Europe is on the decline, with infectious diseases confirmed as one of the causative factors. However, no data on the epizootiological situation of wild fish in the Czech Republic are currently available. In this study, brown trout (n = 260) from eight rivers were examined for the presence of viral and parasitical pathogens. Salmonid alphavirus-2, infectious pancreatic necrosis virus, piscine novirhabdovirus (VHSV) and salmonid novirhabdovirus (IHNV) were not detected using PCR. Cell culturing showed no viruses as well, and serological analysis of 110 sera did not detect any specific antibodies against VHSV or IHNV. Fish from two rivers were positive for the presence of piscine orthoreovirus-3 (PRV-3), subtype PRV-3b. However, none of the PRV-3-positive fish showed gross pathologies typically associated with PRV infections. By far the most widespread pathogen was Tetracapsuloides bryosalmonae which was confirmed in each of the examined locations, with a prevalence of up to 65% and 100%, as established by immunohistochemistry and PCR, respectively. Furthermore, up to 43.8% of fish showed signs of proliferative kidney disease caused by T. bryosalmonae, suggesting that this parasite is a main health challenge for brown trout in the Czech Republic.

Kudoa sp. (Myxozoa, Multivalvulida): first report in five commercial fish species from the Canary Islands-FAO 34 (Macaronesia-Spain).

Kudoid myxozoans have been reported causing serious chronic problems in marine fisheries, by reducing the market value of infected fish through pathological damage to the host musculature. We report here the overall prevalence of a Kudoa species in 84/277 (30.3%) fishes from 20 different species of high commercial value captured between October 2011 and December 2013 from the United Nations Food and Agriculture Organization (FAO) 34 commercial fishing area, near the coast of the Canary Islands (Spain). Macroscopic examination showed myxozoan-like cysts in skeletal muscle from 5 of the 20 fish species examined, with the following prevalences: Pagellus acarne (86.7%), Pagellus erythrinus (46.5%), Serranus cabrilla (27.8%), Spondyliosoma cantharus (19.4%), and Sarpa salpa (28.6%). Infection intensity was determined based on spore counts following muscle tissue digestion. Morphometric studies to characterize the species and DNA sequence analysis results suggest that these infections are attributable to a Kudoa species closely related to Kudoa trachuri. This paper reports the first study on a multivalvulidan species to be identified from the Canary Islands. Furthermore, this is the first report of Kudoa parasites in all of the hosts mentioned above, with the exception of P. acarne.

Hormonal stimulation of carp is accompanied by changes in seminal plasma proteins associated with the immune and stress responses.

Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. This study aimed to compare the proteome of carp seminal plasma between control and Ovopel-induced males using two-dimensional differential in-gel electrophoresis. Ovopel induction increased sperm volume, total sperm count, seminal plasma osmolality, and pH and decreased seminal plasma protein concentration. In total, 36 spots were identified (23 up- and 13 downregulated), corresponding to 23 proteins differentially abundant in seminal plasma after Ovopel induction (p < .05; fold change 1.2). The majority of proteins were associated with the immune and stress responses including the transport protein (hephaestin), antiproteases (fetuin, α2-macroglobulin, TIMP2), complement components (C3, complement factor B/C2A), regulator of the coagulation cascade (plasminogen), modulators of the innate immune response, such as intelectin, ApoA and ApoE, and the cathepsin/cystatin system, and stress response (enolase1). In addition, hormonal stimulation seems to be related to the proteins involved in lipid metabolism, signal transduction, and tissue remodeling. Our results suggest that hormonal stimulation is not just concomitant with the hydration of testis but also induces the synthesis and secretion of seminal plasma proteins involved in sperm maturation and protection against stress induced by administration of the exogenous hormone. SIGNIFICANCE: It is well known that hormonal stimulation of male fish induces the final maturation of spermatozoa. However, molecular and biochemical basis underlying hormone-induced changes in semen is unknown at present. This study for the first time reveals, using proteomic approach, that hormonal stimulation in addition to hydration of testis is accompanied by significant changes in seminal plasma proteins related mainly to immune and stress response, lipid metabolism, signal transduction and tissue remodeling. These changes are associated with gene expression and synthesis and secretion of seminal plasma proteins by reproductive tissues. Overall, our results provide a framework for understanding the molecular mechanism responsible for hormonal stimulation in the reproductive tract of fish males.

Purification, characterization and expression of transferrin from rainbow trout seminal plasma.

Transferrin (TF) is recognized as a multifunctional protein and has been implicated in antioxidative, antimicrobial protection, growth, differentiation and cytoprotection effects. An efficient, original three-step isolation procedure for TF consisting in hydrophobic interaction chromatography, gel filtration and preparative electrophoresis was developed. Rainbow trout TF was found to be N-glycosylated (not O-glycosylated) and phosphorylated at all serine, threonine, and tyrosine residues. The protein consists of several proteoforms with an average molecular weight of 76.9kDa and isoelectric point ranging from 5.2 to 5.7. Rainbow trout TF has two functional iron-binding sites and appears to be quite distinct from carp TF regarding glycosylation and iron-binding properties. The highest gene expression of TF was detected in liver and testis, the lowest was detected in head kidney, spleen and efferent ducts. For the first time TF was identified in the semen of several salmonid species. TF was localized within testis, mainly in spermatozoa, Sertoli, Leydig cells, as well as in both columnar secretory and basal cells within the efferent duct. This work contributes to the existing knowledge information indicating significant variations in TF structure within teleost fish. The results obtained in this study provide valuable data on the TF from trout seminal plasma and the physiological role of this protein in the reproductive tract of salmonids. The results are important for our understanding of the role of TF in the antioxidant protection and resistance to pathogenic infections of reproductive cells. The protective role of TF against environmental pollution with heavy metals, especially during prolonged storage of spermatozoa in the spermatic duct, as well as regulation of spermatogenesis and providing Fe for developing germ cells is also postulated.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

In vitro cytotoxicity and multiplex PCR detection of virulence factors of Yersinia ruckeri isolated from rainbow trout in North West Germany.

The aim of this study was to investigate differences in presence and expression of virulence factors between biotype 1 and 2 strains of 82 Yersinia (Y.) ruckeri isolates, collected from North West Germany during the period of 2004-2012, and to analyze the cytotoxicity of these strains to different fish cell lines. The common virulence factor genes, such as yhlA and yhlB encoding for hemolysin YhlA, rucC and rupG encoding for ruckerbactin, yrp1 and yrpDEF for ABC exporter protein system, and two flagellar genes, including flgA for flagellar secretion chaperones and flhA for flagellar secretion apparatus, were found present in both biotype 1 and 2 isolates of Y. ruckeri collected from North West Germany using multiplex PCR. mRNA expression of these genes was compared between the two biotypes of Y. ruckeri. There was no significant diversity (p > 0.05) in the expression of these genes between biotype 1 and 2 strains. 27 Y. ruckeri isolates from different typing groups were analysed in cytotoxicity tests to common carp brain (CCB), epithelioma papulosum cyprini (EPC), fathead minnow epithelial (FHM) and rainbow trout gonad-2 (RTG-2) cells, respectively. In vitro cytotoxicity of the isolates to CCB, EPC and FHM was higher than that to RTG-2 (p < 0.05). At 15 degrees C the maximum cytotoxicity to FHM and EPC was higher in non-motile strains than in motile stains after an incubation of 24 h (p < 0.05), however, after 48 h, there was no significant difference (p > 0.05) of cytotoxicity between those two biotypes. Our results suggest that biotype 2 strains from North West Germany are homogenous with biotype 1 strains on the basis of genetic virulence factor genes. At lower temperature non-motile Y. ruckeri isolates were found more active than motile strains, which could explain why in winter non-motile strains were found more often responsible for ERM outbreaks than motile strains.

Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.).

Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.

Effects of Enteromyxum leei (Myxozoa) infection on gilthead sea bream (Sparus aurata) (Teleostei) intestinal mucus: glycoprotein profile and bacterial adhesion.

The intestinal myxosporean parasite Enteromyxum leei causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) (Teleostei) that impairs nutrient absorption causing anorexia and cachexia. In fish, as in terrestrial vertebrates, intestinal goblet cells are responsible for the adherent mucus secretion overlying epithelial cells, which constitutes a first line of innate immune defense against offending microorganisms but serves also as substrate and nutrient source for the commensal microflora. The secreted intestinal mucus of parasitized (n = 6) and unexposed (n = 8) gilthead sea bream was isolated, concentrated, and subjected to downward gel chromatography. Carbohydrate and protein contents (via PAS and Bradford stainings), terminal glycosylation (via lectin ELISA), and Aeromonas hydrophila and Vibrio alginolyticus adhesion were analyzed for the isolated intestinal mucins. Parasitized fish, compared with unexposed fish, presented intestinal mucus mucins with a lower glycoprotein content and glycosylation degree at the anterior and middle intestine, whereas both glycoprotein content and glycosylation degree increased at the posterior intestine section, though only significantly for the total carbohydrate content. Additionally, a slight molecular size increase was detected in the mucin glycoproteins of parasitized fish. Terminal glycosylation of the mucus glycoproteins in parasitized fish pointed to an immature mucin secretion (N-acetyl-α-D-galactosamine increase, α-L-fucose, and neuraminic-acid-α-2-6-galactose reduction). Bacterial adhesion to large-sized mucus glycoproteins (>2,000 kDa) of parasitized fish was significantly lower than in unexposed fish.

Effect of hexavalent carcinogenic chromium on carp Cyprinus carpio immune cells.

Chromium is widely used in industrial processes, and is released into aquatic environments by electroplating, tannery and textile industries. Fishes in natural waters or in aquaculture facilities supplied with these waters are exposed to chromium waste and are presumed to be affected by deposits. Herein, we examine the effect of hexavalent chromium on carp Cyprinus carpio derived immune cells. In vitro exposure of carp leukocytes to hexavalent chromium induced cytotoxicity, decreased mitogen-induced lymphocyte activation and phagocyte functions at concentrations between 2 and 200 micromol Cr l(-1). Neutrophils responded to chromium challenge by changes in cell shape together with reduced nitric oxide and reactive oxygen production. This occurred at much lower concentrations than for the cytotoxic effects seen in leukocyte cultures derived from peripheral blood or pronephros. In a similar way, activation of carp lymphocytes by pokeweed mitogen was reduced in a dose-dependent manner, while cytotoxic effects on non-activated lymphocytes were observed at much higher doses of 200 micromol Cr l(-1). Altered lymphocyte and neutrophil functions are considered to be responsible for decreased resistance to pathogens observed in fishes under chronic chromium challenge.