Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method

ABSTRACT:   Bacterial populations in goldfish feces were characterized by the fluorescent in situ hybridization (FISH) method. A total of nine different group-specific rRNA-targeted oligonucleotide probes were used. Approximately half of the microbial cells (57.8 ± 16.7%) were detected with a probe EUB338 and found to be bacteria. The microbial cells in 33–35 of the 35 samples from five specimens strongly hybridized with probes ALF1b, BET42a and GAM42a, suggesting that goldfish intestinal bacteria are mainly composed of α, β and γ-subclasses of Proteobacteria. The fact that a probe AER66 reacted with 25.6 ± 14.2% of the total microbial cells in all 35 samples, demonstrated that genus Aeromonas was the dominant species in the goldfish intestines. Genus Bacteroides including Bacteroides type A detected with a probe BAC303 was observed in 15 of 35 samples while other taxonomic groups determined with HGC69a, CF39a and P72 were detected in 6–11 of 35 samples. These results strongly suggest that Bacteroides shows the greatest daily fluctuation and interindividual variation in the intestines of goldfish. Moreover, the FISH method was proven to be useful for rapid enumeration of taxonomic groups of fish intestinal bacteria.     

Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish. αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.     

Immunological detection of translation products of type V/XI collagen α1 gene and their degradation products in red sea bream muscle

We previously cloned cDNA of type V/XI collagen α1 chain (ColVa1) gene from cultured cells derived from red sea bream embryo. We raised an antibody against the deduced C-telopeptide of ColVa1 in order to detect the translation products of this cDNA and their degradation products in red sea bream muscle. To improve its specificity, the antibody was purified from rabbit antiserum by use of an affinity column cross-linked with recombinant C-terminal peptide of ColVa1 produced by Epicurian coli. The purified antibody recognized a band corresponding to the α chain of type V/XI collagen in western blot analysis of the extract of cultured cells. The antibody also recognized two bands in acid-soluble and pepsin-solubilized collagens, indicating that the translation products of the ColVa1 gene are present in muscle and that bands correspond to α and β chains of type V/XI collagen. A band corresponding to a molecular weight of approximately 65 k was detected in the NaOH extracts of muscle, suggesting that type V/XI collagen α1 chain is restrictedly digested in red sea bream muscle.     

Qualitative and quantitative changes of F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATPase in Japanese flounder and red sea bream associated with rearing temperatures

ABSTRACT:   Fast skeletal muscles of Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major were examined for quantitative and qualitative changes of mitochondrial ATP synthase (F_oF₁-ATPase) in association with rearing temperatures. The specific activities of F_oF₁-ATPase from Japanese flounder reared at 10°C, 15°C and 25°C for 4 weeks were determined to be 81 ± 11, 74 ± 13 and 83 ± 11 nmol/min·mg mitochondrial protein, respectively. The corresponding activity from red sea bream reared at 8°C for 5 weeks was determined to be 65 ± 9 nmol/min·mg mitochondrial protein, which was higher than 33 ± 9 nmol/min·mg mitochondrial protein in fish reared at 23°C. The contents of α- and β-F₁-ATPase in total mitochondrial proteins were not significantly different between fish reared at different temperatures for the two fish species. However, the contents of β-F₁-ATPase in the total fast skeletal muscle extracts, prepared from Japanese flounder reared at 10°C, were 2.1- and 2.9-fold higher than those for fish reared at 15°C and 25°C, respectively. The corresponding content from red seabream reared at 8°C was 2.2-fold higher than that for fish reared at 23°C. Therefore, the changes in F_oF₁-ATPase depending on rearing temperatures were species-specific.     

Gene expression of fatty acid-binding proteins, fatty acid transport proteins (cd36 and FATP) and β-oxidation-related genes in Atlantic salmon (Salmo salar L.) fed fish oil or vegetable oil

Relative gene expression pattern of fatty acid transport proteins (FATP and cd36), intracellular fatty acid-binding proteins (FABP3, FABP10 and FABP11), β-oxidation-related genes [carnitine palmitoyl transferase II (CPTII), peroxisome proliferator-activated receptor β (PPARβ), acyl-CoA oxidase (AOX), long-chain fatty acyl-CoA synthetase (FACS), acyl-CoA dehydrogenase (dehydrogenase)] and uncoupling protein 2 (UCP2) was assessed by RT-qPCR in Atlantic salmon muscle (red and white), liver, heart, myosepta and visceral fat. FABP11, a FABP isoform not previously described in Atlantic salmon, was highly expressed in visceral fat and myosepta and at the lower level in red muscle, white muscle, myosepta and heart. Furthermore, Atlantic salmon were fed either a diet containing fish oil (FO) or a complete replacement of FO with a vegetable oil blend (55% rapeseed oil, 30% palm oil and 15% linseed oil; VO) for the production cycle (27 months from start of feeding and until ∼4.5 kg mean weight). The expression of genes related to β-oxidation, fatty acid uptake and transport in the white muscle indicate ( n  = 3) significant down-regulation in VO fed Atlantic salmon and correlated with previously reported white muscle triacylglycerol stores and β-oxidation. FABP11 in visceral fat and myosepta was also down-regulated in VO fed fish.     

Histochemical, fluorescent and electron microscopical appearance of hepatocellular ceroidosis in the Atlantic salmon Salmo salar L.

Abstract. In this work it is established that a close relationship exists between the feeding of rancid feed (containing high amounts of lipid peroxides and α, β-unsaturated aldehydes) and lipoid liver degeneration (LLD) in fish. The main feature of the disease is a large accumulation of ceroid in hepatocytes, giving positive histochemical staining reactions with a number of selective stains. Tissues also reacted strongly for lipid peroxides and fluoresced a bright yellow-green at 450 nm. This is the first report on the ultra structure of ceroid in LLD. The accumulations consist of bodies containing cholesterol or cholesterol ester crystals and inclusions with membranous profiles. The latter type of inclusion is considered to be representative of ceroid.     

Cryptocaryon irritans recombinant proteins as potential antigens for sero‐surveillance of cryptocaryonosis

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero‐surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans‐infected fish, particularly asymptomatic fish. In this study, we proposed a serum‐based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme‐linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.     

Pharmacokinetics of oxolinic acid in gilthead sea bream, Sparus aurata L.

This is the first study on the pharmacokinetic parameters of oxolinic acid (OA) in gilthead sea bream, Sparus aurata L. The kinetic profile of OA was studied after a single intravascular injection (20 mg kg⁻¹) in 100 g fish at 20 °C. The distribution half-life ( t _1/2α) and the elimination half-life ( t _1/2β) of the drug were found to be short (0.51 and 12.60 h, respectively). The drug penetration from the plasma to the tissues was adequate as the apparent volume of distribution of the drug at steady-state ( V _d(ss)) was found to be 2.11 L kg⁻¹. The mean residence time ( MRT ) of OA was short (14.25 h) and the total clearance rate ( Cl _T) of the drug was low (0.15 L kg⁻¹ h⁻¹). The bioavailability ( F %) of OA following oral administration (30 mg kg⁻¹) was also low (14%). Maximum values were observed for muscle at 0.5 h after injection, with levels declining as with subsequent sampling. At the first two time points (0.5 and 1 h) plasma levels of OA were higher than muscle, however, the reverse was evident for subsequent samples. Following oral administration, highest muscle levels were found at 16 h and, with the exception of the 24-h sampling, muscle OA concentrations were higher than plasma at all time points. The fast elimination of OA suggests short withdrawal times with reference to human consumption of treated fish.     

Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae)

ABSTRACT: A novel sulfotransferase (O-ST), which transferred the sulfate group of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to O-22 of 11-α,β-hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500-fold and the yield was 4%. On affinity chromatography with a PAP-agarose column, O-ST was observed in the bound fraction, and N-ST specific to N-21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O-ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O-ST did not require metal cations for its activity. O-ST required PAPS as the sole source of sulfate. O-ST transferred a sulfate group from PAPS to only O-22 of 11-α,β-hydroxy STX and not to N-21 of these toxins. These observations suggested that two ST, N-ST and O-ST, participate in the sulfation of PSP toxins.