The protective effect of humic-rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.).

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic-rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic-rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite-derived humic-rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic-rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic-rich water from the recirculating system, the synthetic humic acid or the Leonardite humic-rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic-rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.

A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus.

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12-fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed-atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub-sets of twins and normal full-sibs. Consistent with previous reports, the normal embryo sub-set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2-8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA-binding motif protein, X-linked (rbmx), SRY-box containing gene 3 (sox3) and alpha-thalassemia/mental retardation syndrome X-linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X-chromosome inactivation.

Immunisation of fish by bath immersion using ultrasound.

Ultrasonic irradiations (USI) as a means to open routes in the skin, thus facilitating the transdermal delivery of vaccines that will improve the effectiveness of vaccination by immersion, are reviewed in this paper. Based on our recent results in goldfish and carp it could be summarized that: (i) USI significantly improved the antigen uptake and enhanced antibody response; (ii) the requirements for high antigen concentrations, which are needed for simple bath immersion, could be considerably reduced in presonicated fish; (iii) after bath immersion, the antigen was slowly released from the skin to the blood in which its presence could still be detected 24 hours later. This retardation of the antigen in the skin was suggested to be due to a possible interaction with cells of the local immune system, in which it is processed and recognized. It is concluded that the recent advances in biotechnology of immunization with recombinant DNA and the use of DNA vaccines, together with the improvement of their administration using USI, provide interesting prospects for the further application of vaccines against viral and even parasitic diseases of fish.

Influence of visible light and ultraviolet irradiation on motility and fertility of mammalian and fish sperm.

OBJECTIVE: The effects of visible light irradiation on sperm motility, fertility, and reactive oxygen species (ROS) formation were investigated and compared in ram and fish (tilapia). BACKGROUND DATA: Low-energy visible light has previously been found to modulate various processes in different biological systems. In the literature, it is accepted that the first step following visible light irradiation is the formation of ROS by endogenous cellular photosensitizers. METHODS: Sperm of ram and tilapia were irradiated with various light sources (400-800 nm white light, 660 nm red light, 360 nm blue light, 294 nm UV), and their motility and fertility rates were measured. The amount of ROS generated by irradiation was estimated using electron paramagnetic resonance (EPR) technique. RESULTS: Sperm taken from tilapia showed higher motility and fertility following red and white light irradiation. In contrast, the motility and fertility of ram sperm were slightly increased only by red light. A negative effect on motility and fertility of sperm of both species was obtained following irradiation with UV and blue light. The amount of ROS produced in irradiated tilapia sperm was much higher than that of ram sperm. CONCLUSIONS: The results show that different wavelengths differentially affect tilapia and ram sperm motility and fertilization. The difference in response to the various light sources might be explained by the different amounts of ROS formation by ram and tilapia, which are in agreement with the physiology of fertilization appropriate to each of these species. Based on these results, it is suggested that in vitro fertilization in mammals should be performed in darkness or at least under red light.

Evidence for the existence of an IL-2-like lymphocyte growth promoting factor in a bony fish, Cyprinus carpio.

Activity promoting the growth of carp T-like cells has been found in supernatants of mitogen (PHA)- and alloantigen (MLR)-stimulated carp leukocyte cultures. Activity level in culture supernatants was elevated by phorbol myristate acetate. Proliferation of carp T-like lymphoblasts was also promoted in the presence of Il-2-containing supernatants of mammalian origin. The significance of these findings is discussed.

The mixed leukocyte reaction (MLR) in carp: bidirectional and undirectional MLR responses.

Two-way and one-way mixed leukocyte reaction (MLR) was demonstrated in peripheral blood leukocytes (PBL) of carp. Primary two-way MLR in randomly selected donor pairs were highly variable. Weak primary responses could be strongly augmented by mutual in vivo priming of the reacting donors. One-way MLR was performed using irradiated (16,000 R) allogeneic PBL as stimulators. Reciprocal responses of randomly paired donors were usually unequal, suggesting the usefulness of this method for genetic analysis of MLR-recognized histocompatibility antigens in carp. Kinetics of the primary and secondary two-way MLR were studied, as well as the kinetics of primary one-way MLR against pooled allogenic stimulator cells.

Regulatory effect of temperature and antigen upon immunity in ectothermic vertebrates. II. Primary enhancement of anti-hapten antibody response at high and low temperatures.

Enhancement of primary anti-hapten antibody response was more efficient when carp were preimmunized with modified carrier, rather than with its native form, especially when they were challenged with a slightly substituted penicilloyl-BSA conjugate (Pen5 BSA). No significant enhancement was obtained when the fish were challenged with a heavy conjugate (Pen30 BSA). When fish kept at optimal temperature were preinjected with the modified carriers, rising titers of anti-hapten antibodies were obtained even when the fish were transferred to low temperature just before being challenged with Pen5 BSA. It was concluded that modified carriers are more efficient in enhancing the anti-hapten antibody response and light conjugates are needed to make the cell cooperation possible. It was also suggested that when helper memory cell maturation was allowed to develop at optimal conditions, both cell cooperation and antibody synthesis could occur at low temperatures.

The cells involved in the immune response of fish. III. Culture requirements of PHA-stimulated carp (Cyprinus carpio) lymphocytes.

The optimalization of the culture conditions for carp peripheral blood lymphocytes was studied using the thymidine uptake technique. The quantitative and/or qualitative requirements of this culture were determined with respect to the source of serum, the concentrations of 2-ME, glutamine and PHA, and with respect to the initial cell density. High individual variations, shown along the experiments, pointed out the existence of low and high responders to the mitogenic stimulus. These individual variations were considerably reduced and the mitogenic response extremely enhanced by the addition to the culture medium of optimal concentrations of charcoal adsorbed pooled homologous serum (4%), 2-ME (125-250 microM), glutamine (8 mM), PHA (10-40 microliter per ml culture, for the batch used) and by seeding the cells at the optimal density of 2 x 10(5) per microplate well. Finally, the kinetics of cell growth were studied at optimal concentrations of all these factors. The peak response was obtained between days 6 and 7 at 28 degrees C, and the mean doubling time at the exponential phase of the culture, at this temperature, was about 14 hours.

Heterogeneity of mitogen-responsive lymphocytes in carp (Cyprinus carpio).

Peripheral blood leukocytes (PBL) of carp respond in vitro to a variety of phytomitogens, shown to be T-cell specific or B-cell specific in mammalian systems. Some basic differences have been observed in the proliferative response of carp PBL to PHA (phytohemagglutinin), ConA ( concanvalin A) and LPS (lipopolysaccharide): (1) The response to PHA and ConA was found to be highly dependent upon the continuous presence of mitogen in the medium, in contrast to LPS, where after the initial stimulation, cells could continue to proliferate for several days without mitogen. (2) Lymphoblasts grown in long term culture with either PHA or Con A could be transferred into medium containing the other mitogen without impairing cell proliferation, but cell growth was reduced to background level following transfer into LPS-containing medium. LPS grown cells continue to proliferate independently of the mitogen content of the medium. (3) Co-stimulation with LPS+PHA or LPS+ConA results in a synergistic response, while co-stimulation with PHA+ConA results in inhibition of DNA synthesis. (4) Several morphological differences have been observed between cells proliferating in the presence of PHA and those proliferating in the presence of LPS. It is suggested that while the PHA and ConA responsive cells may belong to the same lymphocyte subpopulation, they are distinct from the LPS-responsive subpopulation.

Non-immunological recognition and killing of xenogeneic cells by macrophages. III. Destruction of fish cells by murine macrophages.

Non-immune non-activated murine peritoneal macrophage killed in vitro fish (Cyprinus carpio) PHA-induced lymphoblasts. Addition of PHA and WGA to effector-target cell cultures did not potentiate the killing. This killing (xenolysis) was expressed by non-elicited and thioglycollate-elicited macrophages as well as by macrophages depleted of lymphocytes. It is suggested that mammalian macrophages have a xenolytic potential towards phylogenetically distant species which is analogous to the capacity of invertebrate phagocytes to destroy xenografts.

Specific activity of carp antisera against beta 2-microglobulin (beta 2m) and evidence for a beta 2m homologue in carp (Cyprinus carpio).

Antisera against rabbit and human beta 2-microglobulin were produced in carp (Cyprinus carpio). These antisera were found to be specific for beta 2m, and detected beta 2m epitopes in various vertebrates including guinea-pig, cow, mouse, rat, dog, horse, cat, sheep, goat, parrot, chicken and frog. In addition, these antisera were also inhibited by an extract from oyster. The differences between the reactivity of these two antisera and the ability of the anti-rabbit beta 2m to distinguish between mouse and rat beta 2m's suggested that the carp itself carries beta 2m epitopes. This hypothesis was corroborated by the ability of a variety of mammalian anti-beta 2m antisera to induce a mitogenic response in carp leukocytes. Finally, a rabbit antiserum against dog beta 2m was shown to precipitate a low molecular weight molecule from carp leukocyte extract. This molecule is likely to represent the carp homologue of mammalian beta 2m.

The cells involved in the immune response of fish: II. PHA-induced clonal proliferation of carp lymphocytes in soft agar culture.

Lymphocytes from peripheral blood of carp proliferate in a clonal culture in soft agar, in the presence of phytohemagglutinin, generating several morphologically distinct types of colonies. Cells from colonies developing on the surface of the agar (surface colonies) and cells from colonies developing within the agar (agar colonies) were studied. Several differences were found between cells from the two types of colonies with respect to morphology, ultrastructure and the distribution of cytoplasmic determinants antigenically related to serum immunoglobulin. Colonies were quantitated as a function of the number of cells seeded, in primary cultures of peripheral blood leukocytes and in secondary (replated) cultures of isolated surface colony cells. The numbers of surface colonies and agar colonies in the two systems were comparable. Preferential formation of surface over agar colonies was noted, and there was an initial concentration of cells (individual for each fish) which resulted in optimal colony growth. This method was found to be suitable for isolating highly homogeneous subpopulations of PHA-responsive lymphocytes, which could subsequently be further expanded in liquid culture. A requirement for an exogenously produced growth factor (possibly similar to mammalian Interleukin 2) in the maintenance of long-term clonal cultures is suggested.

The coelomocytes of the earthworm Lumbricus terrestris: morphology and phagocytic properties.

The coelomocytes of Lumbricus terrestris have been classified and described, based on Wright's stained preparations and on living cells. The five major categories consist of basophils, acidophils, neutrophils, granulocytes and chloragogen cells. Both the acidophil and chloragogen cell groups contain two subgroups. Granulocytes also exhibit heterogeneity with respect to staining properties of granules. Some possess acidophilic granules, some basophilic granules, and others contain both types. Granules of acidophils have been observed to be occasionally excreted from the cells. All cell types, with the exception of chloragogen cells, produce pseudopodia and are capable of phagocytosis, a vital component of the earthworm's immune response.

Regulatory effect of temperature and antigen upon immunity in ectothermic vertebrates. I. Influence of hapten density on the immunological and serological properties of penicilloyl-carrier conjugates.

Sodium penicillin was conjugated to sheep erythrocytes and optimal quantities, added to a 5% SRBC suspension, were determined for haemagglutination (12-5 mg/ml) and for haemolysis (50 mg/ml) using carp antibodies and carp complement. The epitope density on the BSA molecule was gradually increased, when increasing amounts of sodium-penicillin were added to a constant quantity of BSA, until a maximum of about thirty penicilloyl groups were bound. Low conjugates, having less than seven haptenic groups per one BSA molecule, were found to stimulate carp for both anti-hapten and anti-carrier antibodies. The higher conjugates having seven and more haptenic groups were found to stimulate carp for anti-panicilloyl antibodies but not for anti-BSA antibodies. A booster dose with native BSA, given to the Pen30 BSA preimmunized carp, gave rise directly to a secondary-like response. In the rabbits, however, both heavy and low conjugates were found to stimulate antibody production for the hapten as well as for the carrier. It was suggested that the modified BSA in the heavy conjugates loses its ability to stimulate B cells, probably due to a decrease in local concentration of antigenic determinants in the BSA molecule, but its ability to stimulate helper cells is not affected for this reason.

Studies on phagocytosis in fish. I. In vitro uptake and killing of living Staphylococcus aureus by peripheral leucocytes of carp (Cyprinus carpio).

The clearance and uptake of Staphylococcus aureus were studied in vitro in whole blood of immunized and non-immunized carp. Bacterial numbers were determined by : (1) counting viable cells in agar plates; (2) counting radioactivity of 14C-labelled bacteria. Similar results were obtained by these two methods. Neither the bacterial growth nor the ingestion of bacteria into the cells seem to be influences by immunization. In both immunized and non-immunized groups the number of intracellular bacteria increased rapidly during the first 120 minutes, then progressed at a slow rate during the next 60 minutes, after which there was a rapid decrease in the number of bacteria only in the immune blood. It is concluded that the phagocytic cells of immune blood are more effective in activating or killing bacteria.

Production of F1 and F 2 diploid gynogenetic tilapias and analysis of the "Hertwig curve" obtained using ultraviolet irradiated sperm.

In this study, a Hertwig effect with a non-typical biphasic curve was obtained using sperm irradiated with increasing intensities of UV. The first phase of the UV curve appeared to be quite different from that normally demonstrated using γ or x-ray irradiation. This difference is characterised throughout the length of the first phase by (1) low and stable embryo hatching rates of about 3.5%, and (2) exclusive formation of haploid embryos at any irradiation intensity. Additionally, at both phases, the ability of the sperm to induce morula formation was not affected at all, and no aneuploidy nor chromosomal fragments could be seen. Therefore, it was suggested that in this fish the lethal effect of UV irradition on sperm is mainly expressed on early differentiative events during embryogenesis, which lead to a degeneration of the embryos during early stages of their development. The possible mechanism by which haploidy is achieved during the first phase is discussed. Two generations of diploid gynogenetic tilapias were induced by activating Oreochromis aureus eggs with UV-irradiated O. niloticus sperm and by using the heat-shock technique, at optimized conditions, for the prevention of the second polar body extrusion. Species specific dominant genetic markers (serum esterases and tail striation) were used to confirm the exclusive content of the maternal genome in gynogenetic offspring. Very low survival rates (0.36%) were shown in F1 gynogenetic fish as well as a high incidence of malformations among survivors. In the second gynogenetic generation, both significantly higher survival rates (3.6%) and a considerably reduced incidence of malformations were obtained. We suggest that low frequencies of recombination occur in this species and cause a rapid increase in the inbreeding level. This is followed by the expression of lethal and defective genes that are considerably reduced after second generation selection.

Sex determination in Sarotherodon (tilapia) : Part 2: the sex ratio as a tool for the determination of genotype - a model of autosomal and gonosomal influence.

The simplest possible model of the sex determination process adding autosomal influence to a minimal number of sex chromosomes was developed to explain matings of Tilapia (Sarotherodon) species. Eighteen different genotypes, each having two autosomes (AA, Aa, or aa) and two sex chromosomes (WX, WY, WW, XY, XX or YY) involved in sex determination, are predicted by the theory. Their sex (10 males and 8 females) were determined using a series of directed graphs, showing the relative strength of the chromosome pairs, developed on the basis of Chen's sex ratio results (Chen 1969). This theoretical model predicts eight different sex ratios (0∶1, 1∶3, 3∶5, 1∶1, 9∶7, 5∶3, 3∶1, 1∶0 ♀∶♂); three of them are not predicted by the WXYZ theory. The greatest part of these sex ratios have been obtained experimentally in extensive series of crosses between related species of Tilapia and their hybrids, carried out by several authors. The theory succeeds in explaining all of Chen's results, including those ratios 5∶3 and 0∶1 seen in certain crosses but not predicted by the WXYZ theory. The importance of the autosomes is seen in comparisons of the genotype pairs (AaWY, aaWY), (AaXY, aaXY) and (AAWW, AaWW) in which the first genotype in each case is male while the second is female as proven by the sex ratio results. The members of the pair differ only in the substitution of one autosome for the other. To test the theory, experiments consisting of hormonal sex reversion and a series of crosses are proposed. Finally, theoretical and practical implications of the theory are discussed.