Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

A Checklist of Metazoan Parasites from Rainbow Trout (Oncorhynchus mykiss)

An extensive literature survey on metazoan parasites from rainbow trout Oncorhynchus mykiss has been conducted. The taxa Monogenea, Cestoda, Digenea, Nematoda, Acanthocephala, Crustacea and Hirudinea are covered. A total of 169 taxonomic entities are recorded in rainbow trout worldwide although few of these may prove synonyms in future analyses of the parasite specimens. These records include Monogenea (15), Cestoda (27), Digenea (37), Nematoda (39), Acanthocephala (23), Crustacea (17), Mollusca (6) and Hirudinea (5). The large number of parasites in this salmonid reflects its cosmopolitan distribution.     

Ubiquitin genes in rainbow trout (Oncorhynchus mykiss)

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.     

Rainbow trout (Oncorhynchus mykiss) brain cells biosynthesize novel docosahexaenoic acid-derived resolvins and protectins-Mediator lipidomic analysis

Docosahexaenoic acid (DHA; C22:6 n-3) is an abundant fatty acid in fish phospholipids. In the present study, we employed liquid chromatography-ultraviolet spectrometry-tandem mass spectrometry and dissociated rainbow trout (Oncorhynchus mykiss) brain cells to determine whether fish utilize endogenous DHA to produce the recently uncovered novel lipid mediators termed the resolvins and protectins, generated by mammalian cells [Serhan CN, Hong S, Gronert K, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 2002; 196:1025-37; Hong S, Gronert K, Devchand P, Moussignac R-L, Serhan, CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. J Biol Chem 2003;278:14677-87]. Trout brain cells biosynthesize a range of recently identified di- and tri-hydroxy-containing bioactive products from endogenous sources of DHA when challenged in vitro. We identified neuroprotectin D1, resolvin D5, resolvin D1 and resolvin D2 from trout brain cells. Each compound was identified on the basis of its characteristic physical chemical properties that included MS, MS-MS, UV spectra and chromatographic behavior. The monohydroxy products from DHA, signatures of DHA conversion by lipoxygenases, were also identified. These included both 14S-hydroxy-docosahexaenoic acid and 17S-hydroxy-docosahexaenoic acid. The biosynthesis of these novel bioactive lipid mediators, namely resolvins and protectins, by fish cells provides the first evidence for the conservation of these structures from fish to humans as chemical signals in diverse biological systems.     

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout.     

Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor

Cystatins are a superfamily of low K_i cysteine proteinase inhibitors found in both plants and animals. Cystatin C, a secreted molecule of this family, is of interest from biochemical and evolutionary points of view, and also has biotechnological applications. Recently we cloned and sequenced the cDNA for rainbow trout (Oncorhynchus mykiss) cystatin C [Li et al., 1998. Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C. Comp. Biochem. Phys. B 121, 135–143]. To explore the relationship between protein structure and function of trout cystatin C, we established a bacterial system for expression of the protein. Trout cystatin C expressed in the cytoplasm of bacterial cells did not have detectable protease inhibitory activity. Activity was regained by Ni–NTA chromatography under denaturing conditions followed by dialysis-based refolding. Titration of purified cystatin C preparations with papain indicated that 20% of the total protein had been converted to the active form after one refolding cycle. Expression levels were 3–5 mg/l. The protease-inhibitory properties of recombinant trout cystatin C were similar to those of human and chicken cystatin C derived from biological sources and recombinant cystatin C derived from rat and mouse genes. The K_i for papain was 1.2×10⁻¹⁵ M, exhibiting the high affinity binding unique to this family of protease inhibitors.     

Investigating Fear in Rainbow Trout (Oncorhynchus mykiss) Using the Conditioned-Suppression Paradigm

Trout learned the operant task of pendulum-pressing for a food-reward in a mean of 4.3 sessions lasting 1 hr. In a separate phase, fish also learned—through classical conditioning—to associate a neutral light cue with an aversive stimulus. When again allowed to pendulum-press for food, after aversive classical conditioning, there was a drop in the rate of responding. The mean rate dropped from 3.6–2.9 responses per min. Most important, when the light-stimulus was superimposed on a steady bout of pendulum-pressing, trout ceased to press the pendulum and did not resume activity until termination of the light-stimulus (mean number of responses during a 3-min interval immediately prior to light-stimulus = 14.3 vs. during 3-min light-stimulus = 0.1). Psychologists have used this decrease in operant responding, or “conditioned emotional response,” as a tool to examine the psychological nature of this type of aversive conditioning. In this study, the fish demonstrated various results under this paradigm similar to those shown by “higher” nonhuman animals, therefore challenging the view of fish as unconscious, nonsentient animals.     

Specificity of antibodies established from mammals in rainbow trout (Oncorhynchus mykiss)

This study deals with the question as to whether antibodies established for mammals are also specific for rainbow trout. The reason for this examination is the major economic importance of rainbow trout in aquaculture and the growing scientific attention. However, there are few primary antibodies so far defined for this fish species. Therefore, the aim of the current study was to test the ability of 15 commercially available antibodies for rainbow trout in an indirect immunofluoresence assay to analyse tissue sections of organs. Five commercially available primary antibodies were identified, which were directed against proteins of one or two organs/ cell types in rainbow trout.     

Gastrointestinal assimilation of Cu during digestion of a single meal in the freshwater rainbow trout (Oncorhynchus mykiss)

Gastrointestinal processing and assimilation of Cu in vivo was investigated by sequential chyme analysis over a 72 h period following ingestion of a single satiation meal (3% body weight) of commercial trout food (Cu content=0.42 micromol g(-1)) by adult rainbow trout. Leaded glass ballotini beads incorporated into the food and detected by X-ray radiography were employed as an inert marker in order to quantify net Cu absorption or secretion in various parts of the tract. Cu concentrations in the supernatant (fluid phase) fell from about 0.06 micromol mL(-1) (63 microM) in the stomach at 2 h to about 0.003 micromol mL(-1) (3 microM) in the posterior intestine at 72 h. Cu concentrations in the solid phase were 10 to 30-fold higher than in the fluid phase, and increased about 4-fold from the stomach at 2 h to the posterior intestine at 72 h. By reference to the inert marker, overall net Cu absorption from the ingested food by 72 h was about 50%. The mid-intestine, and posterior intestine emerged as important sites of net Cu and water absorption and a potential role for the stomach in this process was also indicated. The anterior intestine was a site of large net Cu addition to the chyme, probably due to large net additions of Cu-containing fluids in the form of bile and other secretions in this segment. The results provide valuable information about sites of Cu absorption and realistic concentrations of Cu in chyme fluid for future in vitro mechanistic studies on Cu transport in the trout gastrointestinal tract.     

Wohlfahrtiimonas larvae sp. nov., isolated from the larval gut of Hermetia illucens (Diptera: Stratiomyidae)

A novel, Gram-negative, facultative anaerobic, motile and short rod-shaped bacterium, strain KBL006^T was isolated from the larval gut of Hermetia illucens, Black soldier fly. The 16S rRNA gene sequence of strain KBL006^T showed 96.4 % similarity to that of Wohlfahrtiimonas chitiniclastica S5^T. Strain KBL006^T grew optimally at 30 °C, at pH 8.0 and in the presence of 1–2 % (w/v) NaCl. Oxidase activity and catalase activity were positive. The major fatty acids were C_18:1 ω7c, C_14:0, and C_16:0. The major respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol, and two phospholipids. The G+C content of the genomic DNA was 45.2 mol%. Based on these polyphasic data, strain KBL006^T is considered to represent a novel species in the genus Wohlfahrtiimonas, for which the name Wohlfahrtiimonas larvae sp. nov. is proposed. The type strain is KBL006^T (= KACC 16839^T = JCM 18424^T).     

Analysis of microsatellite variation in the rainbow trout (Parasalmo (Oncorhynchus) mykiss) from Kamchatka

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.