Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication.

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp.

Vaccination of carp against SVCV with an oral DNA vaccine or an insect cells-based subunit vaccine.

We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV.

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.

Oxidative burst and nitric oxide responses in carp macrophages induced by zymosan, MacroGard(®) and selective dectin-1 agonists suggest recognition by multiple pattern recognition receptors.

ß-Glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing ß-glucans, with the C-type lectin receptor dectin-1 in conjunction with Toll-like receptor 2 (TLR2), considered key receptors for recognition of ß-glucan. In our studies to determine the possible involvement of these receptors on carp macrophages a range of sources of ß-glucans were utilized including particulate ß-glucan preparations of baker's yeast such as zymosan, which is composed of insoluble ß-glucan and mannan, and MacroGard(®), a ß-glucan-based feed ingredient for farmed animals including several fish species. Both preparations were confirmed TLR2 ligands by measuring activation of HEK293 cells transfected with human TLR2 and CD14, co-transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. In addition, dectin-1-specific ligands in mammals i.e. zymosan treated to deplete the TLR-stimulating properties and curdlan, were monitored for their effects on carp macrophages by measuring reactive oxygen and nitrogen radicals production, as well as cytokine gene expression by real-time PCR. Results clearly show the ability of carp macrophages to strongly react to particulate ß-glucans with an increase in the production of reactive oxygen and nitrogen radicals and an increase in cytokine gene expression, in particular il-1ß, il-6 and il-11. We identified carp il-6, that was previously unknown. In addition, carp macrophages are less, but not unresponsive to selective dectin-1 agonists, suggesting recognition of ß-glucans by multiple pattern recognition receptors that could include TLR but also non-TLR receptors. Candidate receptors for recognition of ß-glucans are discussed.

Comparative study of ß-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by ß-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by ß-glucans.

The role of physiology in the divergence of two incipient cichlid species.

Sexual selection on male coloration has been implicated in the evolution of colourful species flocks of East African cichlid fish. During adaptive radiations, animals diverge in multiple phenotypic traits, but the role of physiology has received limited attention. Here, we report how divergence in physiology may contribute to the stable coexistence of two hybridizing incipient species of cichlid fish from Lake Victoria. Males of Pundamilia nyererei (males are red) tend to defeat those of Pundamilia pundamilia (males are blue), yet the two sibling species coexist in nature. It has been suggested that red males bear a physiological cost that might offset their dominance advantage. We tested the hypothesis that the two species differ in oxidative stress levels and immune function and that this difference is correlated with differences in circulating steroid levels. We manipulated the social context and found red males experienced significantly higher oxidative stress levels than blue males, but only in a territorial context when colour and aggression are maximally expressed. Red males exhibited greater aggression levels and lower humoral immune response than blue males, but no detectable difference in steroid levels. Red males appear to trade off increased aggressiveness with physiological costs, contributing to the coexistence of the two species. Correlated divergence in colour, behaviour and physiology might be widespread in the dramatically diverse cichlid radiations in East African lakes and may play a crucial role in the remarkably rapid speciation of these fish.

Turbot resistance to Philasterides dicentrarchi is more dependent on humoral than on cellular immune responses.

Philasterides dicentrarchi is a ciliate that causes high mortalities in cultured turbot, Psetta maxima (L.). This pathogen displays high phagocytic activity and after entering the body it multiplies and feeds on host cells and tissue components. In previous studies, we found that complement, activated through the classical pathway, is a potent killer of P. dicentrarchi. Here, we compared the killing activity of turbot leucocytes and humoral factors against two virulent isolates of P. dicentrarchi, in order to determine the importance of leucocytes in the defence against this pathogen. Components of P. dicentrarchi (ciliary and membrane) stimulated turbot leucocytes, and increased the respiratory burst, degranulation and the expression of pro-inflammatory cytokines. We tested the susceptibility of ciliates to reactive oxygen and nitrogen species, by incubating them with different oxidative systems (H(2)O(2), Fe/ascorbate, which induces lipid peroxidation, an O(2)(-) donor (XOD/HX), an NO donor (SNAP) and an ONOO(-) donor (SIN-1)), for 24h. Both isolates were susceptible to high concentrations of H(2)O(2,) Fe/ascorbate, XOD/HX, and SIN-1 but were resistant to incubation with SNAP. Leucocytes became strongly activated when they were in contact with or were phagocytosed by the ciliate. Incubation of P. dicentrarchi with a combination of fresh serum and specific antibodies killed most of the ciliates, but the addition of leucocytes to ciliate cultures did not increase the toxicity to the ciliates. On the contrary, the number of ciliates increased when leucocytes were added to the culture because the ciliates fed on them. Despite being activated, leucocytes did not produce sufficiently high concentrations of toxic substances to kill the parasite. The most virulent isolate was that which induced greatest activation of leucocytes but was least susceptible to complement. We concluded that humoral factors such as complement (activated through the classical pathway) are critical for fish defence against P. dicentrarchi and that cellular responses appear to play a minor role, if any, in defence against this ciliate.

Interaction between the blood fluke, Sanguinicola inermis and humoral components of the immune response of carp, Cyprinus carpio.

The effect of Sanguinicola inermis on serum antibody and complement activity in Cyprinus carpio was assessed using an ELISA and haemolytic assays. Possible immune evasion strategies were assessed using immunodetection of host proteins on the surface of the parasite. Carp acclimatized to 20 or 25 degrees C were infected by exposure to 500 cercariae or injected intraperitoneally with 150 cercariae, and serum monitored over a 63-day period. In cercariae-injected carp, irrespective of time and temperature, a significant increase occurred in complement activity being greatest at 25 degrees C. In addition, fish exposed to the cercariae of S. inermis and maintained at 20 degrees C the level of complement activity was significantly higher after 5 weeks compared to controls. At 20 degrees C intraperitoneal injections of parasites increased serum antibody levels which peaked after 7 days. In contrast, at 25 degrees C, antibody levels were maintained over 63 days. Exposure of fish to infection did not appear to stimulate antibody production. Immunofluorescence studies revealed 'host-like' molecules on the surface of the cercarial body exposed to carp serum and adult flukes obtained directly from the fish or cultured for 24 h in L15 medium. The possible role of 'host-like' molecules in immune evasion is discussed and the response at different temperatures is related to infection dynamics.

Immunization of goldfish with excretory/secretory molecules of Trypanosoma danilewskyi confers protection against infection.

Trypanosoma danilewskyi is a protozoan that lives in blood and other tissues of fish. In the aquaculture industry, economic losses may be substantial, since the prevalence of infection may approach 100% and the parasite may cause significant mortality in farmed carp. Most of the surviving fish acquire resistance after elimination of the primary infection. In this study, we examined whether protection against infection could be induced in naïve goldfish immunized with excretory-secretory (ES) products of the parasite. The ES extracts were administered in conjunction with Freund's complete or incomplete adjuvant (FCA and FIA, respectively). Parameters used to assess the efficacy of immunization after challenge infection, included prevalence of infection, abundance of parasites, and presence of parasite-specific antibodies. Intraperitoneal inoculation of ES products in FCA conferred protection against T. danilewskyi infection (P<0.05). Administration of ES products (with or without FIA) conferred insignificant levels of protection. In an attempt to identify the immunogenic ES molecules, we assessed whether anti-parasite antibodies present in the serum collected from experimentally infected fish or rabbits immunized with ES recognized parasite ES antigens. An immunoblot using rabbit anti-parasite antibody revealed a recognition profile of molecules (two antigens of approximately 102-104 and 70-72kDa) similar to that of immune goldfish serum. While the antigens that confer protection need further molecular characterization, our results suggest that the administration of ES products may allow for the design of control strategies for T. danilewskyi.

Immune modulation by fish kinetoplastid parasites: a role for nitric oxide.

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

Molecular and functional characterization of a fish inducible-type nitric oxide synthase.

Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1,127-amino acid protein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transcription factor NF-kappaB was shown to play a role in the induction of iNOS transcription.

Identification and characterization of a fish natural resistance-associated macrophage protein (NRAMP) cDNA.

The mouse Lsh/Ity/Bcg locus regulates natural resistance to intracellular pathogens, and the Nramp1 gene was isolated as its candidate. Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. In the present study, a full-length cDNA for carp NRAMP has been isolated and characterized. Nucleotide and predicted amino acid sequence analysis indicate that the carp NRAMP encodes a 548 amino acid membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionarily conserved consensus transport motif. The peptide sequence identity among carp and human NRAMP2 is 78%, and 65% with human NRAMP1. Reverse transcription-polymerase chain reaction revealed that carp NRAMP is ubiquitously expressed. Phylogenetic analysis, using neighbor-joining, showed that the carp NRAMP protein clustered together with mammalian NRAMP2 proteins.

Major histocompatibility genes in cyprinid fishes: theory and practice.

The first teleostean MHC sequences were described for carp. Subsequent studies in a number of cyprinid fishes showed that the class I sequences of these fishes are of particular interest. Two distinct lineages (Cyca-Z and Cyca-U) are found in the common and ginbuna crucian carp, but only the U lineage is present in zebrafish and other non-cyprinid species. The presence of the Z lineage is hypothesised to be the result of an allotetraploidisation event. Both phylogenetic analyses and amino acid sequence comparisons suggest that Cyca-Z sequences are non-classical class I sequences, probably similar to CD1. The comprehensive phylogenetic analyses of these sequences revealed different phylogenetic histories of the exons encoding the extracellular domains. The MHC genes were studied in laboratory and natural models. The natural model addressed the evolution of MHC genes in a Barbus species flock. Sequence analysis of class I and class II supported the species designation of the morphotypes present in the lake, and as a consequence the trans-species hypothesis of MHC polymorphism. The laboratory model involves the generation of gynogenetic clones, which can be divergently selected for traits such as high and low antibody response. The role of MHC molecules can be investigated further by producing a panel of isogenic lines.

Immune responses after injection vaccination of fish.

During the last 20 years considerable progress has been made in describing and understanding the immune system of fish. Fish are the phylogenetically oldest vertebrate group with an immune system showing clear similarities to the defence systems of mammals and birds. Both innate immunity (non-specific responses) and acquired immunity (specific responses) are important for the defence against invading pathogens. Antigen injection will evoke humoral and cellular responses, which show the expected characteristics of specificity and memory. Variability in the results can be caused by external factors such as antigen dose, temperature and handling stress. Moreover, the genetic background of the fish may also play a role. The use of standardised inbred fish lines is recommended for the optimal development and evaluation of vaccines and vaccination procedures.

Immunogenetics of disease resistance in fish: a comparative approach.

The study of the genetic regulation of infectious disease resistance depends on the availability of inbred lines or selection lines of the species under investigation. The small numbers of such lines of fish has limited the strategy in teleosts to studies of associations between disease and immune/health traits. Attempts to correlate genetic differences in immune responsiveness with survival after experimental challenge with pathogenic bacteria have failed to define immune parameters that can substantially aid selection for genetic resistance to infectious diseases. Advantages and disadvantages of selection strategies as illustrated by mouse and chicken models are discussed. In this study we summarize the present situation in fish as well as our attempts to develop gynogenetic lines of carp for immunogenetic research.

Characterization of isogenic carp (Cyprinus carpio L.) lines with a genetically determined high or low antibody production.

Antibody production to dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) served as the immune parameter to divergently select carp (Cyprinus carpio L.) to produce high- and low-responder F1 hybrid lines. Antibody production to trinitrophenyl-lipopolysaccharide (TNP-LPS) and to DNP-KLH were similar in magnitude. By contrast, some high-responder lines were low responders to DNP-human serum albumin, and vice versa. Low-responder carp were relatively susceptible to infection with the parasite Trypanoplasma borreli. This suggested that at least one gene with a major influence on resistance differed between the two homozygous parents (69, 85) used to generate the high- and low-responder homozygous families, respectively. The isogenic lines showed no within-line variation in DNA fingerprints, but differed with respect to their MhcCyca-DAB genes.

Multiple regulation of carp (Cyprinus carpio L.) macrophages and neutrophilic granulocytes by serum factors: influence of infection with atypical Aeromonas salmonicida.

Normal carp serum contains inhibitory and stimulatory factors for macrophage and neutrophilic granulocyte respiratory burst activity. As stimulatory factors were only effective in combination with phorbol myristate actetate (PMA) activation, it is concluded that they are probably linked to protein kinase C activation. Both the stimulatory and inhibitory factors are heat stable. Macrophage- and neutrophilic granulocyte-enriched cell fractions from the pronephros of carp had high respiratory burst- and high bactericidal in vitro responses to virulent atypical Aeromonas salmonicida bacteria. Serum factors were inhibitory for the A. salmonicida induced respiratory burst activity. No change in inhibitory or stimulatory serum factors could be observed during a 12-day challenge experiment with A. salmonicida, or during a rechallenge of survivors from a previous sub-lethal infection. The sensitivity of macrophages and neutrophilic granulocytes to stimulation of respiratory burst activity by PMA was not significantly altered. Culture supernatants from PHA pre-treated lymphocytes stimulated the respiratory burst activity of macrophages and neutrophilic granulocytes suggesting that serum factors may partially be lymphocyte derived.

Investigations into the ubiquitous nature of high or low immune responsiveness after divergent selection for antibody production in common carp (Cyprinus carpio L.).

This paper reports on the selection of individual carp with a high or low antibody response, in combination with reproduction by gynogenesis, in order to develop well-characterised inbred carp lines consisting of practically unlimited numbers of carp with the same genotype. Two homozygous progenies, previously characterised as having a high or low immune response to dinitrophenyl keyhole limpet haemocyanin (DNP-KLH), were immunised with either a T-dependent (DNP-human serum albumin (DNP-HSA)) or T-independent (trinitrophenyl lipopolysaccharide (TNP-LPS)) hapten-carrier complex. In comparison with the antibody response after DNP-KLH immunisation, the response to DNP-HSA was observed to be highly variable and did not differ between the divergently selected progenies. This suggests that the divergent selection for antibody production to DNP-KLH has been carrier-specific. Immunisation with T-independent TNP-LPS induced a very rapid response which differed between the high and low responders, and likely measured changes in the DNP-specific precursor pool of B cells caused by the selection. A number of selected individuals with a high immune response to DNP-KLH were infected with Trypanoplasma borreli, a haemoflagellate parasite of carp, to examine a possible relationship between the increase in immune responsiveness and disease resistance, but no change could be detected. However, individual homozygous carp were able to escape inbreeding depression and survive the infection. Such carp would be likely candidates for gynogenetic reproduction to obtain viable inbred carp lines.