Herpesvirus cyprini: a search for viral genome in infected fish by infected fish by in situ hybridization

Abstract. Herpesvirus cyprini (CHV) genome was traced in carp, Cyprinus carpio L., after acute infection by the method of in situ hybridization with biotinylated probes. The viral genome was detected in several tissues including cranial nerve ganglia. Subcutaneous tissue and spinal nerves. However, at this stage, viral antigens were not detected and the virus was not isolated. The viral genome was also detected in the same fish tissues when papillomas were present which contained viral antigens and even infective virus particles. After papilloma regression, the viral genome was still detected in these tissues. It is suggested that CHV becomes latently established in cranial nerve ganglia, subcutaneous tissue and spinal nerves, and is associated with the induction and recurrence of papillomas.     

The fishery of the Ea Kao reservoir, southern Vietnam: a fishery based on a combination of stock and recapture, and self-recruiting populations

The present paper describes the fishery of the Ea Kao reservoir in south Vietnam. Historical data on the total production and total numbers of fish stocked were available from 1983 to 1996, and this information, together with the results of monthly monitoring of the fishery from June 1996 to December 1998, was used in the present study. A number of gears are used in the Ea Kao fishery, the most important being gill, lift and integrated nets, and beach seines. The fishery of Ea Kao is based on the annual stocking of advanced fry/early fingerlings of 0.3–1.0-g bighead carp, Hypophthalmichthys nobilis (Richardson), silver carp, Hypophthalmichthys molitrix (Cuvier & Valenciennes) and rohu, Labeo rohita Hamilton, as well as the self-recruiting indigenous species Toxabramis houdemeri Pellegrin and two exotic species, i.e. Nile tilapia, Oreochromis niloticus (L.), and common carp, Cyprinus carpio L. On average, the regularly stocked species contribute to 78% of total production, which has been around 400–450 kg ha⁻¹ in the last few years. The monthly peak catches of stocked species tend to coincide with the rainy season from May to October and the catches of self-recruiting species peak between February and April. A significant relationship ( P < 0.001) exists between the stocking density in year n (SD, n ha⁻¹) and the yield of stocked fish in year n + 1 ( Y , kg ha⁻¹):     

Herpesvirus cyprini: detection of the viral genome by in situ hybridization

Abstract. A method of in situ hybridization with biotinylated probes is described for the spceific detection of the Herpesvirus cyprini (CHV) genome, both in CHV infected cell cultures and tissue specimens prepared from carp fry infected with the same virus. From molecularly cloned PstI-fragments of CHV DNA, two fragments were selected as probes for in situ hybridization which had no detectable homology, either with DNA of fathead minnow (FHM) cells used for virus propagation, or with the DNA of several fish herpes-viruses. The method of in situ hybridization was based on a system using streptavidin-biotinylated alkaline phosphatase for the detection of hybridization.     

Experimental pathogenicity of reovirus 13p2 for juvenile American oysters Crassostrea virginica (Gmelin) and bluegill fingerlings Lepomis macrochirus (Rafinesque)

Abstract. A viral agent (13p₂), isolated from clinically normal juvenile American oysters ( Crassostrea virginica ) and characterized as a new serotype of reovirus, was tested to determine if it could replicate and produce disease in experimental juvenile oysters. Because the virus replicated well in the bluegill fry (BF-2) fish cell line, fingerling bluegills Lepomis macrochirus were included in the pathogenicity experiments. Exposure of oysters to the 13p₂ virus in ambient seawater resulted in no significant mortality and no increased virus titres or histological lesions. Virus particles were not observed in tissues of exposed oysters when examined with transmission electron microscopy (TEM). Intraperitoneal inoculation of the 13p₂ virus into bluegills resulted in 44% mortality associated with a focal necrotic hepatitis. Evidence of virus replication, when evaluated histological or by virus titration, was observed in 94% of 32 inoculated fish. Samples of infected livers examined with TEM revealed typical cytoplasmic arrays of 13p₂ virus particles in affected hepatocytes. Rising virus titres and hepatic lesions also occurred in bluegills exposed to water containing the 13p₂ virus. These results indicated the natural host of the 13p₂ virus was not the American oyster, but that it was a significant pathogen for at least one fish species.     

Effects of oestradiol-17β or 17α-methyltestosterone administration on gonadal differentiation of largemouth bass Micropterus salmoides (Lacepède)

Monosex populations can be a valuable management tool in culture of larger size largemouth bass (>400 g). In this study, we investigated the effective mode and duration of oestrogen and androgen administrations to produce monosex largemouth bass populations. The experiment consisted of nine treatments. In oral administration groups, we fed 40-day-old fry either 200 mg of an oestradiol-17β (E₂) kg⁻¹ diet or 60 mg of a 17α-methyltestosterone (MT) kg⁻¹ diet for 30, 45 or 60 days. In bath treatments, we immersed fry in a 1 mg MT L⁻¹ solution for 5 h a day on three or six occasions. For control treatment, we fed fry an ethanol-treated diet for 45 days. The frequency of females in the control group was 53.1%. Oral administration of E₂ at all durations resulted in slight increases in the frequency of females (59.8–70.5%). Both modes of androgen administration at all durations were ineffective in altering phenotypic sex. The experimental results of our study indicated that male differentiation passed the point of being completely and functionally influenced by exogenous oestrogens, while female differentiation had already taken place and was no longer responsive to exogenous androgens in 40-day-old (33.5 mm) largemouth bass fry.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

Identification and expression analysis of ghrelin gene in common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

ABSTRACT:   A ghrelin gene has been cloned and sequenced in common carp Cyprinus carpio . Ghrelin cDNA is composed of 461 bp [with a 36-bp 5'-untranslated region (UTR) and a 113-bp 3'-UTR], which translates into a protein of 103 amino acid residues. Carp ghrelin (preproghrelin) contained a predicted signal peptide of 26 amino acid residues, the ghrelin domain ( Gly ²⁷– Val ⁴⁵) and C-terminal peptide ( Gly ⁴⁶– Phe ¹⁰³). Homology analysis of the ghrelin domain of carp with that of other known ghrelin in vertebrates showed good similarity to teleost ghrelin (50–81.8%). Hydropathy analysis based on the deduced amino acid sequence of ghrelin domains in teleosts showed a similar profile. Carp ghrelin clustered with ghrelin of goldfish Carassius auratus and other teleosts, away from mammalian, reptilian, avian, amphibian and chondrichthian ghrelin, by phylogenetic analysis. Genomic organization of carp ghrelin gene was composed of four exons and three introns, which was the same as that of other teleosts and human ghrelin genes. The carp ghrelin gene was expressed in unstimulated tissues such as foregut, hindgut, spleen and brain. In spleen cells, expression of the ghrelin gene increased upon stimulation with lipopolysaccharide (LPS), phytohemagglutinin (PHA) or imiquimod. The identification of carp ghrelin gene and the analysis of the modulation of its expression in immune-activated conditions will allow a more complete analysis of the roles of ghrelin in teleosts.     

Vaccination of channel catfish, Ictalurus punctatus (Rafinesque), by immersion and oral booster against Edwardsiella ictaluri

Abstract. A commercially prepared vaccine against Edwardsiella ictaluri was used to vaccinate 12-day-old channel catfish fry by immersion, or by immersion plus an oral booster 2 months later. One month after the fish were fed the booster vaccine, they were challenged by waterborne exposure to 2·1 × 10⁶ cells ml⁻¹ of E. ictaluri. Immersion only vaccinated fish suffered 6·7% mortality and immersion plus oral-boosted fish had a 3·3% mortality. Mortality among non-vaccinated controls was 96·7% and was significantly ( P < 0·01) above the vaccinated mortality. The relative per cent survival for the immersion-only fish was 93·1, while it was 96·6 for the immersion plus oral-boosted fish. Agglutinating antibody titres of the vaccinated fish were significantly ( P < 0·05) higher than the control fish. When the ponds were drained 6 months after stocking, 42·7% of non-vaccinated, 56·3% of immersion-only and 70·8% of immersion plus oral-boosted fish were harvested. Survival of immersion plus orally-boosted fish was significantly ( P < 0·05) higher than the controls of immersion-only fish. Duplicate populations of immersion plus oral-booster-vaccinated fish grew 34% and 56% faster, respectively, on an average daily gain than the control fish, while immersion-only fish in one pond grew 20% less per day and fish in the second pond grew 48% faster.     

Effects of Seasonal Variations, Thyroid and Steroid Hormones, and Carp Pituitary Extract on the Artificial Production of Channel Catfish Ictalurus punctatus× Blue Catfish I. furcatus Hybrids

The primary objective was to compare thyroid hormones and levels of carp pituitary extract for the artificial production of female Ictalurus punctatus × male I. furcatus hybrid catfish. The effects of different carp pituitary extract dosage rates (5, 6, 9, and 10 mg/kg), carp pituitary extract (6 and 10 mg/kg) supplemented with the thyroid hormones thyroxine (T₄) and triiodothyronine (T₃), or pregnenolone (DHP) were determined for inducing ovulation of female channel catfish, fertilization of channel catfish eggs with male blue catfish sperm, and hatching rate of these embryos. Hormone treatments thyroxine and tri-iodothyronine with carp pituitary extract, carp pituitary extract alone, and pregnenolone with carp pituitary extract used to artificially produce hybrid catfish were not different in terms of ovulation rates, eggs/kg, fry/kg body weight of female channel catfish, fertilization rates, or hatching rates (P > 0.05). These findings suggest that lower amounts of carp pituitary extract may be used to induce spawn of female channel catfish for production of channel-blue catfish hybrids and the addition of thyroid and steroid hormones is ineffective at the rates used in this study.     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

Localization of Zn-binding protein in the digestive tract tissue of common carp

ABSTRACT: The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn-binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na⁺/K⁺-ATPase (82%), organic phosphate (90%) and the Zn-binding protein (79%), but only part of the 5'-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn-binding protein, Na⁺/K⁺-ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the 'membrane-like' portion of the submucosal and muscular layers. It is proposed that most of the Zn-binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.     

Polysaccharide-induced protection of carp, Cyprinus carpio L., against bacterial infection

Abstract. The efficacy of 10 polysaccharides (curdlan, inulin, krestin, laminaran, lentinan, levan, schizophylian, selerogiucan, yeast glucan and zymosan) to enhance protection of carp, Cyprinus carpio L., against bacterial infection was investigated. Carp were intraperitoneally injected with the polysaceharides (2–l0 mgkg^-1) on days 1 and 4, and challenged with Edwardsiella tarda on day 7. Among the polysaccharides tested, lentinan, schizophyllan and scleroglucan, which are l,6-branchcd-β-l,3-glucans, significantly increased the survival rate. They also induced a protective effect against Aeromonas hydrophila at a dose of 5 mg kg^-1. The ability of the polysaccharides to activate the alternative complement pathway (ACP) was examined by incubating the polysaccharides with carp serum and measuring the residual ACP activity. At a final concentration of 0.l mgml^-1, l,6-branched-β-1,3-glucans greatly reduced (76–77%) the ACP activity. Therefore, it is suggested that the protective effect of the l,6-branched-β-1,3-glucans may be associated with the activation of ACP.     

Effect of sudden increase in discharge in a large river on newly emerged Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) fry

Abstract– Downstrcam displacement of salmonid fry due to flow increase, from 12–15 m³/_s to >100 m³_s, was documented in the river Suldalslåen, Western Norway. In May only fry of brown trout ( Sulmo trutta ) occurred in the drift, while from the beginning of June only newly emerged Atlantic salmon ( Sulmo sular ) were found. The maximum number of Atlantic salmon fry drifting during a single day was estimated to be 17 000 individual. Their density in the drift was higher during the night than during the day, and their appearance in the drift coincided with the predicted period of emergence. Total brown trout numbers in the drift were estimated to vary between 4000 and 16 000 per day. Fry displaced downstream from the lowermost part of the river were lost from the population. The total losses were estimated to be between 75 000 and 100 000 Atlantic salmon fry which represents between 5.6 and 11.1%) of juvenile mortality during the first year of life.     

Fry age and size effects on immersion immunization of brook trout, Salvelinus fontinalis Mitchell, against infectious pancreatic necrosis virus

Abstract. Brook trout, Salvelinus fontinalis Mitchell, fry were divided into eight age groups of 1–8 weeks post-hatch (wph) and immunized by a single direct immersion in formalin-inactivated infectious pancreatic necrosis virus ( ipnv ). After a direct-immersion ipnv challenge given 4 weeks later, only fry immunized at 2, 3 and 6wph showed protection. The relative per cent survival 60 days after ipnv challenge was highest in 2 and 3 wph fry (45–50%) and decreased as fish increased in age or size. The same response was obtained one year later when four age groups of fry, from 1 to 4wph, were immunized with a different serotype and dose of ipnv . The 2 and 3 wph fry had mean weights between 49 and 60mg at immunization. Killed vaccines administered by immersion have not previously been reported as inducing protection in salmonid fry of such low weights. Analysis of the growth of the fry suggests that protection against IPNV requires immunization in the eleutheroembryo phase, during the time of slow weight gain. This appears to be in direct contrast to the stage of ontogeny and weight growth rate required for successful immunization against the bacterial pathogen Vibrio anguillarum. Although immunization with two ipnv serotypes reduced mortalities from challenges with these same virus isolates, it did not prevent ipnv infection of fry in any age group.     

Induced diploid gynogenesis and polyploidy in crucian carp, Carassius auratus gibelio (Bloch), x common carp, Cyprinus carpio L., hybrids.

Abstract. Results of a 16 year (1977–1992) study of hybrids obtained from crossing two cyprinid fish, crucian carp, Carassius auratus gibelio (Bloch) (bisexual form), and common carp, Cyprinus carpio L., are presented. The ability to produce diploid eggs in F₁ females (resulting from chromosome endoreduplication in early oogenesis) was responsible for the high yield of spontaneous diploids in gynogenetic progenies of F₁ females and for triploidy in hybrids from back-crosses of F₁ females with males of the parent species. Different types of hybrids, including diploid and triploid gynogenetic, tetraploid (amphidiploid) and androgenetic progenies, were obtained. Diploid and some triploid hybrids (females only) were reproduced inter se by induced gynogenesis in several subsequent gynogenetic generations. Amphidiploid hybrids were obtained by crossing triploid F₈ females with diploid common carp males or by crossing diploid gynogenetic females with diploid inverted hybrid males producing diploid sperm. Results of cytogenetic investigations and fish culture properties in hybrids are presented. The scientific and practical significance of the data obtained are discussed.     

Sex reversal and growth of Oreochromis spilurus (Günther) in brackish and sea water by feeding 17α-methyltestosterone

Abstract. An investigation was undertaken to study the effect of 17α-methyiteslosterone (17-MT) on the sex reversal and growth of tilapia, Oreochromis spilurus (Günther), in brackish water (3–5 ppt) and sea water (40 ppt). Seven experiments were performed; four in brackish water and three in sea water. Yolk-sac fry (1 or 3 day age) were immersed (7 or 24 h/day) in water containing hormone at a concentration of 0–10mg/l for 4–25 days. The swim-up fry were subsequently fed either normal or hormone-impregnated diets (30–70 mg/kg) for another 35 days starting from first feeding. The growth and SGR of fry were better in groups which received hormone both in water and food as compared with water or food alone. Immersion In 5–10 ppm (24 h/day) brackish water solution for 5 days did not bring any change in growth or sex ratio. However, immersion in sea water solution for 25 days (7 h/day) at a concentration of 1 ppm induced positive growth but no change in sex ratio. Higher doses either did not affect or reduced growth and SGR but induced higher percentages (77–83%) of males. Complete masculinization (100%) was observed only in brackish water when fish were immersed in 2·5 ppm solution for 4 days (24 h/day) and later fed 50 mg/kg in diets for 35 days. These results were further confirmed when the immersion schedule was modified to 10 days (7 h/day) and feeding 50–70 mg/kg hormone. It can be concluded from these studies that in brackish water complete masculinization of O. spilurus is possible through immersion in 5·0 ppm for a minimum of 75 h followed by feeding with 50 mg/kg food for a minimum of 40 days starting from first feeding.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Evaluation of carbohydrate rich diets through common carp culture in manured tanks

Four diets (T₀–T₃) were formulated reducing the fishmeal (Indian) component by 100 g kg^–1 from 300 to 0 g kg^–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg^–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m^–2 in mud bottomed cement tanks (18 m²), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P  > 0.05). PER was lowest for the 300 g fishmeal kg^–1 diet treatment (diet T₀), increasing with decrease in dietary fishmeal content (diets T₁–T₃). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T₂ and T₃. The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed.     

Changes in the nutritional parameters of muscles of the common carp (Cyprinus carpio) and the silver carp (Hypophthalmichthys molitrix) following environmental exposure to cyanobacterial water bloom

The present study evaluated the effect of naturally developing cyanobacteria on the composition of muscles of two commercially important freshwater fish species. Fish were exposed to cyanobacterial biomass including Microcystis aeruginosa and Microcystis ichthyoblabe for 4 weeks. Then, they were transferred to dechlorinated potable water without any cyanobacteria for another 4-week period, thus modelling their preparation for consumers. Samples of muscles were collected every week during exposure and subsequent stay in dechlorinated potable water. The cyanobacterial water bloom of 3.9–6 × 10⁵ cells mL⁻¹ (133–383 μg g⁻¹ of total MC DW) induced statistically significant effects only in the content of fatty acids ( P <0.05; P <0.01) in the common carp ( Cyprinus carpio ), while all studied parameters including the content of dry matter and fat ( P <0.01), proteins ( P <0.05), fatty acid composition ( P <0.05; P <0.01) and some amino acids ( P <0.05) were affected in the silver carp ( Hypophthalmichthys molitrix ). This study has shown that cyanobacteria in the environment of commercially produced fish may decrease the dietetic value of fish muscles.     

Effects of Environmental Sodium Chloride on Percent Hatch, Yolk Utilization, and Survival of Channel Catfish Fry

Abstract.— Only limited research has addressed the effect of salinity on hatching of channel catfish Ictalurus punctatus eggs, and no studies have evaluated the effect of salinity on fry development and survival. This study was undertaken to determine the effect of environmental sodium chloride (0, 1, 2, and 4 g/L. NaCl) on percent hatch, yolk utilization, and survival of channel catfish fry. Experiments were conducted in recirculating systems using seven egg masses (1–2 d old). Each egg mass was divided into smaller portions which remained undissociated or were dissociated with sodium sulfite (NaSO₃). Eggs were incubated until hatching. Wet and dry weights were obtained for sacfry at 1 and 5 d post-hatch to determine wet weight gain and dry weight loss, and fry were sampled 7 d after initiation of exogenous feeding to determine survival. Percent hatch, yolk utilization, and survival of fry hatched from undissociated eggs were greatest at 1 g/L NaCl. In addition, treatment of eggs with NaSO₃ significantly reduced percent hatch at all NaCl levels. Although our results indicate that addition of NaCI to hatchery water supplies can increase production of channel catfish fry, additional research is needed before this practice can be recommended on a commercial basis.