Cellular responses of the skin of carp (Cyprinus carpio) exposed to acidified water

The skin of carp was examined after exposure to acidified water. Degenerative cells were common in the upper epidermal layers. During the first days most of these cells exhibited signs of necrosis. Later on the incidence of necrosis decreased and that of apoptosis increased. In the acid-exposed fish, the upper filament cells and pavement cells produced secretory vesicles of high electron density, some of which showed peroxidase activity. This enzyme activity was also present in the glycocalyx covering these cells, and in the cytoplasm of apoptotic cells. Mitotic figures and newly differentiating mucous cells were common in the outer epidermal layers. Mucous cells became elongated and produced mucosomes of high electron density. Mucosomes with peroxidase activity were also found. Club cells increased in number. Chloride cells and solitary chemo-sensory cells, not seen in the controls, appeared in the upper epithelial layer. The skin was invaded by many leucocytes and by pigment-containing cytoplasmic extensions of melanocytes. Some leucocytes apparently penetrated into the club cells. These structural observations reflect the complexity of the physiological response of the skin to acid water.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

Immunoreactive methionine-enkephalin in the caudal neurosecretory system of the carp,Cyprinus carpio

Summary Methionine-enkephalin (Met-enk) was detected by immunocytochemistry and radioimmunoassay in the caudal neuro-secretory system of the carp Cyprinus carpio. Some cells showing urotensin I (UI)-immunoreactivity reacted to Met-enk antiserum, but others did not. Neurons with urotensin II (UII)-immunoreactivity did not react to Met-enk antiserum; neurons with both UI and UII immunoreactivities also displayed a negative Met-enk reaction. Met-enk was detected by radioimmunoassay in the urophysis, although the content was relatively small compared with that found in other parts of the central nervous system and in the pituitary.     

Function of the cytoskeleton in cells with microridges from the oral epithelium of the carp Cyprinus carpio

Superficial cells of the oral mucosal epithelium in the carp and the cytoskeleton of the epithelial cells are examined by scanning and transmission electron microscopy. Microridges are formed on the surface of the epithelium. Epithelial cells contain two types of vesicles: mucous secretory vesicles and coated vesicles. Most of the mucous vesicles are situated in the center of the cell near the Golgi apparatus. In freeze-fracture replicas, intramembranous particles are abundant in the membranes of the secretory vesicles but rare in the apical plasma membrane. Coated vesicles are situated in the apical and subapical cytoplasm. A great number of thick filaments, considered to be keratin filaments, run randomly throughout the cell to form a meshwork. Thick filaments, which are sparse in the central cytoplasm, are connected to the membranes of the secretory vesicles and other membranous organelles. A layer of closely packed thin filaments, considered to be actin filaments, is found just beneath the apical plasma membrane. Microtubules also occur in the apical cytoplasm and run almost parallel to the cell surface. Both kinds of vesicles are connected to the thin and thick filaments. Their functional significance in the regulation of membrane at the free surface is discussed.     

Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses caused by the ectoparasite Ichthyophthirius multifiliis

Real time quantitative PCR (RQ-PCR) assays were developed for the measurement of differential real-time expression of immune-related genes in skin and whole blood from Cyprinus carpio during an infection with the ectoparasite Ichthyophthirius multifiliis. The target genes included the chemokines CXCa and CXCb, the chemokine receptors CXCR1 and CXCR2, the pro-inflammatory cytokines interleukin 1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) and the enzymes inducible nitric oxide synthase (iNOS) and arginase 2. The strongest up-regulation in skin was observed in the IL-1beta, CXCR1 and iNOS genes at 36-48h post-exposure to theronts. A significant up-regulation of the genes CXCa and TNF-alpha was also observed. An up-regulation of the expression of the genes CXCa, CXCR1, IL-1beta and iNOS was likewise found in blood, although the increase in the expression levels was more moderate and the expression peak was detected earlier in comparison with the skin. In addition, CXCR2 and the arginase 2 genes were specifically induced in blood. Our results confirm the role of CXCR1 and IL-1beta as two prominent molecules involved in the initiation of the inflammatory process in fish in relation to an ectoparasite infection. Moreover, this study confirms the role of carp skin as an important source of pro-inflammatory molecules as well as an active modulator of the local inflammation. Finally, expression and regulation of the evaluated genes in blood confirm the important role of the migrated leucocytes in the immune response against I. multifiliis.     

Microridges of oral mucosal epithelium in carp,Cyprinus carpio

Summary The surface of carp oral mucosa is characterized by various patterns of microridges about 0.3 m wide, 0.1 m high, and of various lengths. To elucidate the derivation and function of these microridges, the oral epithelium was examined by light- and electron microscopy. Microridges were present only on the surfaces of the superficial cells. Therefore, microridges on renewed superficial cells are presumed to be formed after old superficial cells have been discarded, and the various patterns of microridges found on the cell surface appear to indicate the progress of their development. In thin sections, the outer leaflet of the plasma membranes of microridges stained strongly with ruthenium red, and the underlying cytoplasm was packed with many fine filaments. The superficial cells contained many secretory vesicles that were PAS-positive but Alcian blue-negative at pH 2.5 and pH 1.0. However, after sulfation the vesicles gave a positive reaction with toluidine blue. These vesicles are secreted by exocytosis at the free surface of the cells. After release, the membranes of the vesicles are thought to be utilized for formation of microridges. On the basis of these observations, the possible function of microridges is discussed.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Life history patterns of the common carp,Cyprinus carpio,in the estuary of the Guadalquivir river in south-west Spain

We examined 1567 carp caught in rice field drainage canals in the estuary of the Guadalquivir river (37° N, 6° 25 W). The stock was comprised of a small number of age groups, among which 0+ and 1+ were dominant. Growth was divided in four periods: rapid (June–July); slow (August–November), no growth (December–March), and resumed growth in April when an annulus was formed. A classification based on 12 wild carp populations revealed that growth depends on latitude, and that growth of the studied stock was mediocre, probably due to high salinity. A decrease in somatic condition coincided with an increase in gonadosomatic index (GSI). Specimens of the same stock, age and/or length showed considerable difference in development of the gonads. Gonadal activity began in September and by late Autumn some gonads had reached up to 20% of body weight. The situation remained static during Winter till April, when most reproduction occurred. Later, GSI values decreased to reach a minimum in September.Maximum average GSI values in females were twice those in males. Both sexes achieved maturity during the third growing season (2+ age-group) at a minimum fork length (F.L.) of 110 mm. Fecundity, before reproduction, was expressed by Fec = 241.701 F.L. (mm) – 26776.2. The sex ratio did not differ significantly from 1 : 1.     

Structural and expression analyses of two vitellogenin genes in the carp, Cyprinus carpio

We cloned and sequenced two vitellogenin (vg) cDNAs of the carp, Cyprinus carpio, using a cDNA library constructed from estradiol-17β (E₂)-treated livers. One was a novel, longer 5000 bp-long cDNA termed vg-B2 encoding 1624 amino acids in a single open reading frame. The other was a shorter cDNA (vg-B1), identical to that registered previously as carp vg cDNA in the international nucleotide sequence database. The deduced amino acid sequences of these two molecules were well-aligned with known vertebrate Vgs sharing common characteristics such as N-terminal lipovitellin I (LVI), phosvitin (PV) and C-terminal lipovitellin II (LVII). The novel Vg-B2 bore a highly conserved GL/ICG motif within the LVII region, in contrast to the shorter Vg-B1 that has a truncated C-terminal and lacks the β-component within the LVII region including the GL/ICG motif. Both vg-B2 and vg-B1 genes were expressed in the livers of females and E₂-injected males. Western blot analysis using anti-Vg and anti-vitellin (Vn) antisera demonstrated that both Vg-B2 and Vg-B1 were detected as polypeptides with an estimated molecular mass of 180 kDa and 160 kDa, respectively, in the blood of females and E₂-injected males. The results suggest the potential utilization of these genes as sensitive xenoestrogenic markers.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Cellular responses of the skin and changes in plasma cortisol levels of trout (Oncorhynchus mykiss) exposed to acidified water

The skin structure and the plasma cortisol levels of trout, Oncorhynchus mykiss, were examined during 7 days of exposure to water of pH 5. By day-4 and-7, the thickness of the epidermis was significantly (P<0.05) less in acid exposed fish than in controls, and degenerative cells were common in the upper epidermal layers. Many epidermal cells exhibited signs of necrosis, and by day-7 many apoptotic cells were also present. Secretory vesicles of high electron density were abundant in the filament cells of the 3–4 outermost layers of epidermis, and intercellular spaces had increased. Mitotic figures occureed throughout the epidermis, with the exception of the outermost cell layer. Mucous cells became elongated after day-1, and later, newly differentiating mucous cells could be seen close to the skin surface, and many mucocytes contained mucosomes of high electron density. Rodlet cells were occasionally seen. Chloride cells appeared similar to those of control fish. Many leucocytes, mainly macrophages and lymphocytes, had penetrated the epidermis via the highly undulating basal lamina, and at day-7, numerous apoptotic lymphocytes were found. In the dermis, melanosomes became dispersed in the cytoplasmic extensions of melanocytes which were present in the epidermis of all acid-exposed fish. Iridocytes were rate after day-4, while fibroblasts were abundant and secreted large amounts of collagen. After 1 day of exposure to acidified water, a significant (P<0.05) elevation of the plasma cortisol level had occurred, but this subsequently declined, and had returned to control values by day-7. The changes in skin structure, however, remained throughout the whole exposure period.     

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis,with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5–ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5–ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

Carotenoids in fish. XXII. Changes in carotenoids in Cyprinus carpio L.

The author investigated the presence of various carotenoids in the different parts of the body of Cyprinus carpio L. by means of columnar and thinlayer chromatography.The material in the form of the fertilized eggs, week-old fry, month-old fry, four-months old fry, one-year old fry and marketable carp were investigated.Yugoslavian carp brought to Poland as fry, and hybrid the wild carp crossed with of the cultivation carp, were also taken for analysis. The carotenoid content of Cyprinus carpio bred in waters polluted by municipal wastes and specimens suffering from diseases of the airbladder was also determined.The content of carotenoids, the provitamin of vitamin A, in Cyprinus carpio depends of many factors existing during the cultivation of these fish.     

The sequential restoration of plasma metabolite levels,liver composition and liver structure in refed carp,Cyprinus carpio

Effect of realimentation was studied on the structure and function of liver tissue of carp,Cyprinus carpio. Yearling carp, after a 3-month starvation period, were renourished at a feeding rate of 1% body weight per day. Samples were taken at refeeding days 0, 1, 2, 5, 22 and 78. Analyses were made of blood metabolites, liver RNA, DNA, lipids, glycogen and protein and of liver enzyme activities. Additionally, liver cytology was examined by means of qualitative and quantitative electron microscopy. The early refeeding period (up to day 5) was characterized by a fast recovery of plasma metabolite concentrations (protein, total lipids, free fatty acids, glucose), a drastic augmentation of hepatic glycogen reserves, and a pronounced increase of total liver weight and liver-somatic index. Constant values of total hepatic DNA showed that liver weight augmentation was not due to cell proliferation, but to a pronounced enlargement of the existing hepatocytes. Major hunger-related structural modifications of carp hepatocytes such as enlarged mitochondria or prominence of the lysosomal compartment were reversed. A significant volume increase of cell nuclei, together with a particularly strong elevation of hepatic RNA concentrations during initial realimentation suggest an immediate stimulation of protein synthesis. Since the cisternae of the endoplasmic reticulum were not reconstituted during that early phase, protein synthesis may have been executed mainly by free ribosomes. With prolonged realimentation, the volume of the endoplasmic reticulum as well as total and relative contents of liver soluble protein continuously increased, whereas RNA concentrations decreased again. An enforcement of liver oxidative capacity was indicated by the augmentation of cellular number and volume of mitochondria. The activities of the enzymes glucose-6-phosphate dehydrogenase and malic enzyme, which convert excess energy into NADPH, increased steadily. Concomitantly, hepatic lipid accumulation was enhanced. In conclusion, liver metabolism during the early recovery phase seems to be dominated both by repair processes and by intensive protein and glycogen synthesis. The liver slows down these processes during prolonged refeeding and directs an increasing percentage of energy and metabolites toward the generation of reducing equivalents and lipid reserves.Abbreviations BW body weight - ER endoplasmic reticulum - FFA free fatty acids - G6PDH glucose-6-phosphate dehydrogenase - LSI liver somtic index - LW liver weight - ME malic enzyme Presented in part as poster abstract at the International Congress on Research in Aquaculture: Fundamental and Applied Aspects. Antibes, France, 6–10 October, 1991     

Fasting and refeeding in carp,Cyprinus carpio L.: the mobilization of reserves and plasma metabolite and hormone variations

Summary Carp, Cyprinus carpio, were subjected to a short term of fasting (2 months) and 12 days of refeeding. The early changes produced in plasma metabolites and hormones (insulin and glucagon) and their respective energy contribution in liver and muscle during fasting and refeeding was studied. Two phases of fasting were differentiated. The first phase (until day 8 of fasting) was characterized by a reduction in the hepatosomatic index mainly due to glycogen mobilization. A transitory increase in plasma glucose and lactate suggested an initial increase in energy demand. No changes were produced in the percentage of glycogen and protein in muscle, but musculosomatic index and the total body muscle protein decreased. Although the most depleted tissue in this phase was the liver, the loss of energy content of total muscle was higher. Stabilization of liver glycogen content, plasma glucose and lactate levels, decreased muscle protein levels and a reduction in the rate of body weight loss characterized the second phase (from day 8 of fasting). Protein content in whole muscle decreased by 22%, similar to the first phase. The energy expenditure of both liver and muscle was lower in this phase. Plasma insulin levels decreased two-fold and plasma glucagon three-fold in the first phase and remained low in the second phase of fasting. Twelve days of refeeding produced a greater increase in daily growth rate than in the control group and a recovery of plasma insulin, glucagon and glucose levels. Liver completely recovered. In contrast, musculosomatic index, protein and lipid content indicated that muscle did not completely recover from the 2 months of fasting, although and overshoot of muscle glycogen was observed.Abbreviations ANOVA analysis of variance - bw body weight - D1, D2, D5, D8, D19, D50 1, 2, 5, 8, 19 and 50 days of fasting, respectively - GSI gonadosomatic index - HSI hepatosomatic index - MSI musculosomatic index - P-DNA deoxyribonucleic acid phosphorus     

Physiological advantages may contribute to successful invasion of the exotic Cyprinus carpio into the Xingyun Lake, China

Feeding and growth traits of Cyprinus carpio and Cyprinus pellegrini (both at age-0) were compared in three experiment, in an attempt to analyze potential causes for the displacement of the native C. pellegrini in the Xingyun Lake, Yuxi, Yunan, China. Experiment I was conducted in water which fluctuated between 15 and 20°C. Experiment II and III were conducted in a laboratory and water temperature was maintained between 20°C and 25°C, respectively. Three common trends were noted for all three experiments: (1) feeding rate of C. carpio was lower than that of C. pellegrini, and this difference was found to be significant in experiment I; (2) growth rate of C. carpio was higher than C. pellegrini, and the difference was found to be significant in experiment II; (3) food conversion efficiency and energy retention efficiency for C. carpio were higher than those of C. pellegrini, and significant differences were noted in experiment I and II. Since the growth period for fish in the Xingyun Lake generally occurs when water temperatures are between 15 and 25°C, it can be suggested that C. carpio has advantages over C. pellegrini in growth and food utilization efficiency, and lower food consumption than C. pellegrini. These physiological traits of C. carpio might allow this species to be more resistant to food shortage and predation, and may be partially responsible for the displacement of C. pellegrini by C. carpio.     

Developmental stages and localization of peroxidatic activity in the leucocytes of three teleost species (Cyprinus carpio L.; Tinca tinca L.; Salmo gairdneri Richardson)

Summary The ultrastructural localization of peroxidase (PO) in the leucocytes of three teleosts (Cyprinus carpio L., Tinca tinca L., Salmo gairdneri R.) has been investigated using the 3,3-diaminobenzidine method. In the heterophilic granulocytes the granules show a species specific structure and are PO-positive at pH 7.6. They can be traced back to small granules arising near the Golgi apparatus (GA) in the promyelocyte. They coalesce to form larger granules and gradually change into the mature type. Myelocytes contain small unreactive granules, and these represent a second granule population. Eosinophils contain one PO-positive granule type (at pH 9), and these granules show a varying density during cell maturation.Basophils are present only in the Cyprinid species, and contain unreactive granules originating from precursors displaying a weakly positive reaction at pH 7.6. The active secretory organelles (RER, GA) are PO-negative, except for a weakly positive reaction in the flocculent matrix of the inner G-cisternae.In promonocytes and monocytes the granules are unreactive, but in the macrophages PO-positive staining occurs in a few small to medium sized granules, and in large vacuoles. At least some of these latter are apparently derived from phagolysosomes containing digested erythrocytes. Thrombocytes and lymphocytes are unreactive.The successive development of PO-positive and negative granule populations in the heterophils, and the PO-reactivity of eosinophils and basophils, show some similarities to the corresponding cells in higher vertebrates, but an analogous PO-positive (azurophil) granule type in monocytes seems to be absent.     

Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.