Isolation of side population cells from ginbuna carp (Carassius auratus langsdorfii) kidney hematopoietic tissues

Side population (SP) cells, characterized by a specific Hoechst dye efflux pattern by flow cytometry were isolated from kidney hematopoietic tissues of ginbuna carp (Carassius auratus langsdorfii). The hematopoietic activity of SP cells was evaluated by the repopulation and multilineage potential using an in vivo transplant system of ginbuna carp (donor) and ginbuna-goldfish hybrids (recipient). In a flow cytometric (FCM) analysis, a small and distinct population of ginbuna SP cells displayed efficient effluxes of Hoechst 33342 was virtually identical to the efflux observed in mammalian SP cells. The frequency of the ginbuna SP cells was 0.17+/-0.08% in the kidney hematopoietic cells. Morphologically, SP cells were composed of small lymphocyte-like cells having a thin-layered cytoplasm and a round nucleus. These characteristics of ginbuna SP cells were very similar to those of mammalian SP cells. Since cyprinid fish have two hematopoietic sites, the head (anterior) and body (posterior, trunk) kidney, the distribution of SP cells were examined in head and body kidney. The proportion of SP cells were 0.33+/-0.15% in the body kidney, but near 0% in the head kidney. After the ginbuna SP cells were injected into ginbuna-goldfish hybrids, the major types of donor-derived cells (erythrocytes, neutrophils, basophils, monocytes, thrombocytes, T and B lymphocytes) were detected in the recipient blood over a long period (9-16 months post-transplantation). In ginbuna carp, SP cells reside in the body kidney and contain primitive populations of hematopoietic stem cells (HSCs).     

The graft-versus-host reaction (GVHR) in the ginbuna crucian carp, Carassius auratus langsdorfii

A model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids was employed to demonstrate the presence of graft-versus-host reaction (GVHR) in a teleost fish. Tetraploid scale grafts on triploid clone members evoked an acute rejection, whereas the reverse transplants were accepted. When sensitized triploid cells were injected into tetraploid recipients, a typical GVHR was induced, leading to death of the recipients within one month. The onset of illness appeared about one week after cell injection as a loss of appetite and constipation, followed by a scale protrusion, severe haemorrhage, local destruction of the ventral skin and prominent splenomegaly. GVHR was most effectively induced by head-kidney cells and peripheral blood leukocytes (PBL), followed by spleen and thymus cells. Donors had to be sensitized at least twice by scale grafting to induce the reaction. A considerable number of recipients injected with cells from donors which had been sensitized by allogenetically different tetraploids died, suggesting a limited polymorphism or heavy cross-reactions between the alleles of the histocompatibility antigens. Ploidy analyses revealed that donor cells greatly increased in the host liver and spleen, constituting approximately 30% of total cells after 2 3 weeks. Most of these features of acute GVHR observed in this fish system are quite similar to those found in mammals and birds. thereby suggesting the presence of allo-reactive cytotoxic T lymphocytes in teleosts.     

Long-term hematopoietic reconstitution by transplantation of kidney hematopoietic stem cells in lethally irradiated clonal ginbuna crucian carp (Carassius auratus langsdorfii)

The study of hematopoietic stem cells (HSCs) has been facilitated by the transplantation of bone marrow cell populations into lethally irradiated mice. It is widely known that HSCs have the capacity for long-term and multilineage hematopoietic reconstitution in lethally irradiated hosts. Here, we developed a transplantation model system using clonal ginbuna crucian carp (Carassius auratus langsdorfii) that were exposed to a lethal dose of X-rays. The minimum lethal dose (MLD) of ginbuna was approximately 25Gy, which is lethal due to hematopoietic failure. The transplantation of kidney hematopoietic cells into lethally irradiated ginbuna resulted in the rescue of recipient fish for more than 180 days. We examined the reconstitution activity of head kidney (HK), body kidney (BK), and spleen cells. Transplantation experiments showed that only HK and BK cells had the long-term and multilineage reconstitution activity. These results indicate that teleost HSCs have the ability to fully reconstitute the hematopoietic system in lethally irradiated hosts, and that they are present in HK and BK, but not in spleen. This transplantation model system using clonal ginbuna is useful for studies of in vivo kinetics and functions of HSCs in teleosts.     

Demonstration of hematopoietic stem cells in ginbuna carp (Carassius auratus langsdorfii) kidney

A model system of ginbuna carp (clonal triploid fish, Carassius auratus langsdorfii) and ginbuna-goldfish hybrids (tetraploid fish, having three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish) was employed to demonstrate the presence of hematopoietic stem cells (HSCs) in kidney hematopoietic tissues. Kidney hematopoietic cells were obtained from ginbuna carp (S3n, donor) and injected into a ginbuna-gold fish hybrid (S4n, recipient) via the caudal sinus. The percentage of S3n cells in the S4n blood was measured by flow cytometry by the difference of DNA content from 3 to 18 months after the injection. Donor-derived major types of blood cells including erythrocytes, thrombocytes, neutrophils, basophils, monocytes, T and B lymphocytes were detected in the recipient blood over a long period (9-18 month posttransplantation). Moreover, recipient lympho-hematopoietic organs such as head kidney, body kidney, spleen, and thymus were reconstituted by donor cells. These results indicate that long-term multipotent repopulating HSCs reside in the donor kidney.     

Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyrinus carpio), goldfish (Carassius auratus) and tench (Tinca tinca)

High spontaneous anti-trinitrophenyl (TNP) activities were found in three Cyprinid fish species: Carp (Cyprinus carpio), Goldfish (Carassius auratus) and Tench (Tinca tinca). The molecules involved, isolated by affinity chromatography on dinitrophenyl-lysine Sepharose (DNP-lysine-Sepharose), had the main characteristics of a high molecular weight immunoglobulin (IgM-like). Affinity measurements were performed on natural anti-DNP/TNP antibodies isolated from nine individual tench sera, using the inhibition of DNP-T4 bacteriophage inactivation technique. The antibodies analysed were more specific for TNP than for DNP. No activity was found against paranitrophenyl hapten. Affinities were all very low, even for TNP. In the three species, natural anti-DNP/TNP antibodies constitute as much as 11 to 16% of the total immunoglobulin concentration. This high level of nitrophenyl-binding serum immunoglobulins either suggests the existence of a particular regulatory mechanism in fish or reflects a generally low antibody diversity in these species.     

Differential expression of vasa RNA and protein during spermatogenesis and oogenesis in the gibel carp (Carassius auratus gibelio), a bisexually and gynogenetically reproducing vertebrate.

The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alphaVasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of vasa and its protein changes dynamically during oogenesis. The alphaVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, Cagvasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alphaVasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation.     

Isolation and partial characterization of immunoglobulins of goldfish (Carassius auratus) and carp (Cyprinus carpio)

Two related species of teleost fishes, the goldfish (Carassius auratus) and the carp (Cyprinus carpio), responded to injection of protein antigens by producing antibodies which resembled the γM immunoglobulins of other vertebrates in size and polypeptide chain structure. No other immunoglobulin classes were observed in these fish. Since the ancestors of the teleosts diverged from those of the tetrapods over 300 million years ago, the present results are consistent with the hypothesis that light chains and μ-type heavy chains emerged early in vertebrate evolution.     

Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.     

Biology and host response to Cyprinid herpesvirus 3 infection in common carp

Viruses from the family Alloherpesviridae form an aquatic clade of herpesviruses infecting fish and amphibia. Diseases caused by these herpesviruses are of increasing importance because of the high morbidity and mortality associated with the infection, and the difficulties in diagnosing latently infected carriers. Cyprinid herpesvirus 3 (CyHV-3) induces a severe disease and mortality in common carp and thus greatly affects carp aquaculture and trade. This review summarises advancements in the understanding of the infection process and the current knowledge on immune responses of carp to CyHV-3. A focus is laid on host genetics and immunity responsible for resistance/survival from the disease and on the viral mechanisms accountable for evasion of carp immune responses. As current knowledge of immune responses to CyHV-3 is still limited, perspectives for future studies are outlined. Analysing CyHV-3 fish-host interactions will be useful and thought-provoking for a basic understanding of fish immune responses.     

Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress, dexamethasone, and metals

The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cuvieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids.