Immune response of juvenile common carp (Cyprinus carpio L.) exposed to a mixture of sewage chemicals.

Pharmaceuticals and household chemicals are important components of municipal sewage. Many of them are biologically active, disrupting not only hormonal regulation of aquatic animals but also, indirectly, disturbing their immunological protection. In the environment, chemicals rarely act as individual substances, but as elements of mixtures. Therefore, the aim of this study was to check whether the acute laboratory exposure of common carp juveniles to a mixture of ibuprofen, sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and 17 α-ethynylestradiol in increasing concentrations, modifies the levels of innate immunity (lysozyme, C-reactive protein) as well as general stress (metallothioneins, heat shock proteins HSP70) markers in brain, liver, gills, spleen and mucus. The levels of the markers were measured by an immunodetection technique. Not only do the pharmaceuticals and household chemicals impair immunological reactions of young carp in various tissues but also do that in a concentration-dependent manner in the liver, gills, spleen and mucus. This has a very important implication, since it may result in higher sensitivity of young fish to pathogens due to energy allocation to defence processes. The comparisons of the pattern of stress reactions in the studied organ samples indicated that mucus appeared to be a good, non-invasive material for monitoring of environmental state and fish conditions.

Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with ß-1,3/1,6-glucan.

The effect of dietary ß-glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard® , a ß-1,3/1,6-glucan, or a ß-glucan-free diet. Fourteen days after feeding, half of the carp from each group were intubated with 109 colony-forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT-PCR-DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with ß-glucan compared to carp from the control group. In ß-glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the ß-glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with ß-glucan may provide protection against infections of the gut in carp.

A full-body transcriptome and proteome resource for the European common carp.

BACKGROUND: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies. RESULTS: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data. CONCLUSIONS: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

Stress-induced adaptation of neutrophilic granulocyte activity in K and R3 carp lines.

Both in mammals and fish, stress induces remarkable changes in the immune response. We focused on stress-induced changes in the activity of neutrophilic granulocytes in the R3 and K lines of common carp, which showed differential stress responses. Our study clearly demonstrates that a prolonged restraint stress differentially affects the activity of K and R3 carp neutrophils. In the K line, stress decreased the respiratory burst, while in the R3 line it reduced the release of extracellular DNA. Surprisingly, the stress-induced changes in ROS production and NET formation did not correlate with changes in gene expression of the inflammatory mediators and GR receptors. In neutrophilic granulocytes from K carp, gene expression of the stress-sensitive cortisol GR1 receptor was significantly higher than in neutrophils from R3 fish, which will make these cells more sensitive to high levels of cortisol. Moreover, upon stress, neutrophilic granulocytes of K carp up-regulated gene expression of the anti-inflammatory cytokine IL-10 while this was not observed in neutrophilic granulocytes of R3 carp. Therefore, we can hypothesize that, in contrast to R3 neutrophils, the more cortisol sensitive neutrophils from K carp respond to stress with up-regulation of IL-10 and consequently reduction of ROS production. Most probably the ROS-independent NET formation in K carp is not regulated by this anti-inflammatory cytokine. These data may indicate a predominantly ROS-independent formation of NETs by carp neutrophilic granulocytes. Moreover, they underline the important role of IL-10 in stress-induced immunoregulation.

Activity of the hypothalamus-pituitary-interrenal axis (HPI axis) and immune response in carp lines with different susceptibility to disease.

The stress response transmitted by the HPA axis is one of the best examples of neuroendocrine-immune interactions that are critical for survival. Analogous to the situation in mammals, the stress response in fish is characterized by the activation of the hypothalamo-pituitary-interrenal axis (HPI). Effects of cortisol on the fish immune system comply with findings in mammals and suggest that the differences in sensitivity to stress will influence the immune response and as a consequence of survival. Therefore, we studied the stress response and its immunity-related effects in four different carp lines (R3, R3xR8, K and R2) that display a differential pathogen susceptibility. Previous studies indicate that R3xR8 and R3 carp are susceptible to bacterial and parasite infection, while R2 and K are relatively resistant to infection. Interestingly, the most striking effect of stress on leukocyte composition and activity was observed in the pathogen-resistant K carp, even though no robust changes in gene expression of stress-involved factors were observed. In contrast, R3 carp showed no spectacular stress-induced changes in their immunological parameters with concurrent significant activation of the HPI axis. Upon stress, the R3 carp showed up-regulation of crf, pomc and gr2 gene expression in the hypothalamus. Furthermore in R3 carp, at all levels of the HPI axis, stress induced the highest up-regulation of il-1ß gene expression. Although we are aware of the complexity of the interactions between stress and pathogen susceptibility and of the risk of interpretation based on correlations, it is noteworthy that the fish more susceptible to infection also exhibited the highest response to stress.

The influence of dietary ß-glucan, PAMP exposure and Aeromonas salmonicida on apoptosis modulation in common carp (Cyprinus carpio).

The association between ß-glucan (MacroGard®) supplemented feed and apoptosis in immune-related organs of common carp (Cyprinus carpio) was studied using fluorescence microscopy and real-time PCR. In addition the effect of Aeromonas salmonicida, LPS and Poly(I:C) injections on this relationship was evaluated. Whilst acridine orange staining revealed that apoptosis levels were independent of MacroGard® and LPS/Poly(I:C) administration or their combination, it was shown that injection with A. salmonicida increased the percentage of apoptotic cells irrespective of the feeding regime. It was apparent that in all the treatments gene expression profiles displayed organ and time dependency. For example no effect was observed at 7 days of MacroGard® administration while 25 days of feeding led to increased iNOS expression and differential up-regulation of anti- or pro-apoptotic genes depending on organ. This may indicate differences in NO sensitivity. MacroGard® also led to an elevation of pro- as well as anti-apoptotic genes in LPS or Poly(I:C) injected fish, while LPS/Poly(I:C) alone had little effect. A. salmonicida caused enhanced iNOS expression and it is possible that the type of apoptosis pathway induced is organ dependent as Caspase 9 is induced in mid-gut but not in pronephros. These results indicate that MacroGard® feeding alone or in combination with other pathogenic factors did not induce significant apoptosis in immune organs.