Synthesis of platelet activating factor by tissues from the rainbow trout, Salmo gairdneri

In mammals, platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator with biological activity at concentrations in the subnanomolar range. Although PAF is known to have many activities in mammals, little is known about its synthesis and importance in other vertebrate groups. We demonstrate here the synthesis of PAF from [3H]acetate by slices of trout gill, kidney, liver and spleen. PAF synthesis was stimulated by the calcium ionophore A23187 and was time-dependent. The radiolabeled PAF produced was characterized by TLC, HPLC, derivatization and by saponification and phospholipase A2 hydrolysis. These findings suggest that PAF may be an important mediator in fish.     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl- -free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA(-) MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.     

B cell maturation factor: effects on various cell populations

B cell maturation factor (BMF) is a lymphokine that promotes the maturation of resting murine splenic B lymphocytes, and the analogous B cell tumor line WEHI-279, to the state of active immunoglobulin (Ig) secretion. All subsets of normal B cells examined, including neonatal and adult B cells, B cells from various organs, and B cells from CBA/N mice, are inducible by BMF. Induction of Ig secretion is independent of thymus-derived cells, LPS receptors, and MHC haplotype, because nude, C3H/He, and mice of many strains are equally responsive to BMF. Purified B cells prepared by using a fluorescence-activated cell sorter also respond to BMF, showing that BMF directly interacts with and triggers Ig secretion by B cells. In limiting dilution cultures, most normal resting splenic B cells or WEHI-279 B tumor cells are inducible by BMF. By using the WEHI-279 cells as a model system, specific aspects of the BMF response have been analyzed. In terms of the degree of stimulation observed, the primary mechanism for the induction of Ig secretion by BMF is an enhanced and balanced synthesis of Ig heavy (H) and light (L) chains. A less significant component of the induced Ig secretion is an increase in the ratio of secretory to membrane H chains produced. Kinetically, the shift in the ratio of secretory to membrane H chain forms occurs first, and this is followed by the increased synthesis of both L and H chains. Responding B cells also die during this induction process. Although the changes in the ratio of H chain forms, H and L synthesis, and cell viability take several days to occur, BMF will program significant later responses after only 1 or a few hr of interaction with target B cells.     

The effects of chronic immune stimulation on muscle growth in rainbow trout

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and Western blotting were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish.     

1,25-dihydroxyvitamin D3 induces CCR10 expression in terminally differentiating human B cells

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.     

Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri)

1. Total blood volume and relative blood volumes in selected tissues were determined in non-anesthetized, confined rainbow trout by using 51Cr-labelled trout erythrocytes as a vascular space marker. 2. Mean total blood volume was estimated to be 4.09 +/- 0.55 ml/100 g, or about 75% of that estimated with the commonly used plasma space marker Evans blue dye. 3. Relative tissue blood volumes were greatest in highly perfused tissues such as kidney, gills, brain and liver and least in mosaic muscle. 4. Estimates of tissue vascular spaces, made using radiolabelled erythrocytes, were only 25-50% of those based on plasma space markers. 5. The consistently smaller vascular volumes obtained with labelled erythrocytes could be explained by assuming that commonly used plasma space markers diffuse from the vascular compartment.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Enzyme activities of plasma and selected tissues in rainbow trout Salmo gairdneri Richardson

This paper describes the determination in rainbow trout of the normal levels in the plasma of eight enzymes known to be significant in animal pathology. Some relationships between plasma enzyme levels and the enzyme activities in selected tissues are discussed. For LDH which is ubiquitously distributed, we chose, by way of saturation tests, the optimum concentrations of pyruvate and NADH in the assay medium. The LDH 'isoenzyme ratio' was determined for heart tissue, liver, and white muscle. When blood was withdrawn from caudal vessels, we observed a net increase of plasma enzyme activities, mainly of CPK and LDH, which was demonstrated to originate from surrounding muscle. Thus cardiac sampling was the only suitable way of obtaining blood for this kind of study. Slightly haemolyzed blood appeared suitable for enzyme determinations except for Alk Pase which is about 50 times more concentrated in erythrocytes than in plasma. CPK was highly concentrated in the heart and the muscle, GOT was concentrated in the heart while the liver appeared to be a valuable source of GDH (as well as the kidney) and GPT.     

Differentially expressed proteins in rainbow trout adipocytes isolated from visceral and subcutaneous tissues

In rainbow trout, subcutaneous (in dorsal and ventral positions) and visceral fat deposits are known to influence the yield of edible flesh, whilst their respective roles in metabolism, storage and release of fatty acids have not, so far, been directly studied. The present work aimed to identify, by using 2D electrophoresis, proteins differentially expressed in isolated mature adipocytes originating from these various localizations in prepubescent females. A total of nine proteins were estimated to be differentially expressed according to the localisation of the adipocytes. Seven protein spots were considered to be present in the three fat deposits at differing abundances, and among them only six were estimated as being specific to fat tissues. Among these, five were more abundant in subcutaneous adipocytes of both sites compared to perivisceral adipocytes. Four were identified: three as H-FABP, ATP synthase, serum deprivation-response protein, indicating higher metabolic activity in subcutaneous adipocytes, while the latter, annexin, indicative of a higher proportion of less mature adipocytes, as also suggested by their smaller mean diameter. The more abundant protein in visceral isolated adipocytes is actin, known to be involved in cytoskeleton structure and to increase during adipogenesis. This allows us to suggest their more mature stage of development, in relation with their higher mean diameter.     

GABA-immunoreactive neurons in the photosensory pineal organ of the rainbow trout: two distinct neuronal populations

Summary The distribution of putative GABA-ergic neurons in the photosensory pineal organ of the rainbow trout was investigated by use of a specific antiserum against -aminobutyric acid (GABA). GABA-immunoreactive (GABA-IR) neurons were located in the rostral portion of the pineal end-vesicle, presumably constituting a population of interneurons. GABA-IR neurons were also found in the pineal stalk. The axons of these neurons were traced along the pineal stalk toward the brain. The terminal areas of these axons could not be established. GABA-IR glial cells were observed in the pineal end-vesicle, but not in the pineal stalk.     

Branched chain alpha-keto acid dehydrogenase in tissues from rainbow trout (Salmo gairdnerii)

Mitochondria isolated from skeletal red muscle of rainbow trout (Salmo gairdnerii) have remarkably high activity of branched chain alpha-keto acid dehydrogenase as measured with alpha-keto isocaproic acid as substrate. The activity of the dehydrogenase increases several-fold after preincubation of the mitochondria with an uncouple and oligomycin. The accumulation of the first product, isovaleryl-CoA is strongly stimulated by high concentrations of CoASH. In the crude mitochondrial lysates we have used, an unknown compound accumulates with time, during incubation with alpha-keto isocaproic acid, CoASH and NAD. The compound, probably a CoA derivative, is more hydrophobic than isovaleryl-CoA.     

Induced gynogenesis in the rainbow trout: Sex and survival of progenies production of all-triploid populations

Summary Gynogenesis could be an efficient way for producing inbred lines in commercial fish species. Gamma-irradiation of sperm gives haploid embryos that all die without hatching; in the present study, we optimized heat treatment of the eggs, in order to produce high rates of diploid gynogenetics. When the eggs are heated to 26 °C for 20 min after 25 min of development, 80% of the embryos hatch, and all the resulting fry are diploid; nevertheless, high mortalities are recorded until feeding start. The monosex female constitution of gynogenetic offspring confirms the female homogamety in the rainbow trout.When the eggs are treated with the same heat shock 25 min after a fertilization with functional sperm, alltriploid populations are obtained; their survival until feeding start is not different from the control.     

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.     

Estimation of introgression in cutthroat trout populations using microsatellites

Introgressive hybridization, mediated by anthropogenic activity, poses a threat to numerous and diverse taxa. The management of introgressed individuals or populations within species of conservation concern is currently the subject of scientific and political debate. We investigate the utility of 10 non-diagnostic microsatellite loci for investigating admixture from introduced Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (O. mykiss) within 25 putative Rio Grande cutthroat trout (O. c. virginalis) populations. We apply five different approaches (correspondence analysis, maximum-likelihood assignment tests, an admixture estimator based on allele frequencies, an admixture estimator based on coalescent theory and an admixture estimator implementing a Bayesian method) and use two alternative O. c. virginalis reference samples. All approaches were capable of identifying one population that consisted entirely of introduced O. c. bouvieri, and three out of five approaches enabled us to discriminate those populations with relatively high levels of non-native introgression from those populations with little or none. Actual estimates of admixture coefficients within a test population, varied, however, with the approach and reference sample used. These results have important implications for policies dividing populations into different management categories according to the estimated proportion of non-native genetic material that they contain.