Licentiatavhandling (sammanfattning) Karlstad : Karlstads universitet, 2008. / Härtill 2 uppsatser.

Thesis (M.A.)--Boston University / The morphology of the cellular constituents of the blood of Salmo trutta was investigated. Blood cell counts and differential counts were included. Cellular elements of the blood were found to be nucleated erythrocytes, lymphocytes, neutrophils, monocytes and thrombocytes. Dried smears of blood which had been stained with Wright's stain indicated the erythrocytes were flat elliptical cells . However, the phase microscope revealed the biconcave shape of the mature erythrocyte, the concavity being interrupted by the central nucleus. Average cell rreasurements were 16.5 microns in length and 10.2 microns in width. [TRUNCATED]

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This study investigated the biological activity of a macrophage activating factor (MAF) produced by activated lymphocytes from the rainbow trout (Oncorhynchus mykiss) and attempts to discover its molecular source. Peripheral blood lymphocytes were shown to release factors with MAF activity following incubation with a variety of stimulants and were subsequently shown to activate macrophages using at least two different methods, the nitroblue tetrazolium (NBT) colourimetric assay and the luminol-dependent chemiluminescent assay. The latter technique detected an immediate response which decayed over a 40 minute period on the addition of cell-free supernatants from activated lymphocytes to macrophages. A number of molecular approaches, including degenerate PCR primer amplification, DNA cross-hybridisation and cDNA library screening were used in this study to try to isolate any cytokine genes from Oncorhynchus mykiss. As a control β-actin cDNA was successfully amplified from Oncorhynchus mykiss using primers based on the salmon sequence. The Oncorhynchus mykiss orthologue of IFN-y was initially targeted. However, although a PCR product of the appropriate size was amplified using degenerate primers based on mammalian and avian IFN-y sequences, the sequence was not related to IFN-y or any other known Oncorhynchus mykiss sequence. A similar strategy was used to try and amplify the Oncorhynchus mykiss orthologue of mammalian IL-15. Again despite amplification of a DNA fragment of approximately the correct size there appeared to be no relationship between it and the known IL-15 sequences. As an alternative strategy a cDNA library from stimulated peripheral blood lymphocytes (PBLs) was constructed and screened using cDNA probes derived from stimulated and non-stimulated PBLs in order to detect mRNAs which might have been upregulated as a result of in vitro stimulation. A number of positive clones were obtained from the differential screening of the library including cDNAs showing similarity to other unidentified fish sequences as well as to a number of proteins predicted to be involved in regulation of cell proliferation, neocorticogenesis and embryo development. Additionally. the library was also screened using ovine cytokine cDNA probes. although no positively hybridising clones were obtained. The ovine IFN-y gene was also used to probe genomic DNA from Oncorhynchus mykiss. but unlike previous studies with human IFN-y gene no hybridisation between the ovine IFN-y gene and Oncorhynchus mykiss DNA was observed. This investigation highlights the potential difficulties of using various molecular strategies such as DNA cross-hybridisation or PCR techniques for the cloning of fish cytokine sequences. Consequently, future strategies for cloning fish cytokine genes may require targeting the biological activity through expression libraries.

M.Sc. (Zoology) / Please refer to full text to view abstract

A knowledge of the basic biology of any fish is a primary requirement for the practical management of that stock of fish. This investigation was directed at a pure culture population of Yellowstone cutthroat trout, to describe the basic biology and provide a basis for management and further research. Kiakho Lake has a surface area of 67.42 acres, a maximum depth of 32 feet and a mean depth of 16.5 feet. Due to the rocky substrate, lack of littoral development and low total dissolved solids, the production of plankton and bottom fauna was small and characteristic of oligotrophic conditions. The food of cutthroat trout in Kiakho Lake in May was comprised of 83.9 percent by volume and 81.3 percent by occurrence of chironomid pupae. In June the food was 46.7 percent by volume and 45.8 and 35.5 percent by occurrence of chironomid larvae and Gammarus respectively. In July the Gammarus were 57.8 percent by volume and 60.3 percent by occurrence. In Lumberton Reservoir and Monroe Lake the Gammarus comprised 51.0 and 55.6 percent by volume and 34.4 and 78.2 percent by occurrence respectively of the food. In Garcia Lake, Chaoborus was 32.9 percent by volume and 36.0 percent by occurrence and the redside shiner, Richardsonius balteatus, was 27.8 percent by volume and 31.8 percent by occurrence. The fish appeared to be second in preference to Chaoborus. The body-scale relationship is described by a straight line having a slope of 1. A graph of instantaneous growth rate plotted against length, revealed that faster growing fish have a faster decrease in growth rate. Due to the absence of certain characteristics e.g. a concavity in the upper limit of the graph, the growth of Kiakho Lake cutthroat appeared to support the view that faster growing fish are selected by the fishery, and that it can be demonstrated in this type of graph. The data, fitted to a Parker and Larkin (1959) growth equation gave a z value of 0.71. The absence of "Lee's Phenomenon" gave support to the premise that the phenomenon can result from selection by a fishery, and invalidated the other ideas concerning the causes as far as this population was concerned. The spawning run in Kiakho Lake was estimated at 3,000 fish. A tagging program revealed that the fish spent on the average of 13 days to spawn, and that there was approximately a 54 percent mortality. The male fish appeared on the spawning grounds first. The female fish showed a decrease in size, later in the run, which was not shown by the males. The eggs hatched sometime in mid June and the young fish apparently spend one year in the outlet stream. The female fish mature between the ages of 2—4 and the males between 1—3. The mean number of eggs per female, plus or minus two standard deviations was 944± 393.29. A multiple regression analysis revealed that body length affected the number of eggs produced, 2.5 times as much as egg diameter. Recommendations were made, due to the probable effects of competition, that cutthroat trout be kept in pure culture populations. It was further suggested that cutthroat trout numbers be maintained in view of the severe reduction and almost extinction of the species in other areas. / Science, Faculty of / Zoology, Department of / Graduate

Differences in food habits and spatial distribution of sympatric Dolly Varden (Salvelinus malma) and cutthroat trout (Salmo clarki clarki) in a small coastal lake were documented by Andrusak (MS 1968). Segregation was inferred to be of the interactive type hypothesized by Nilsson (1965, 1967). The object of this study was to describe feeding behavior of individuals from these sympatric populations, and to evaluate the importance of food exploitation to the segregation process. Individual and paired fish were studied in the laboratory throughout the spring, summer and autumn. The different food habits were found to be due to a number of basic behavioral and morphological differences between the species. Dolly Varden oriented to and rested on the bottom. Cutthroat rested in the water column and were frequently surface oriented. Searching behavior differed between the species. Dolly Varden swam faster and at relatively constant rates. They sampled "mouthfuls" of substrate as they searched. Trout alternately hovered and cruised, sampling specific items. At low light intensities they were much less successful than the char at finding benthic food items. The mouth of the Dolly Varden is small and "scoop-like" compared to that of the cutthroat, and seems particularly well adapted for benthic feeding. Dolly Varden searched persistently for benthic organisms in the absence and presence of surface insects. Cutthroat rapidly switched from bottom to surface feeding if insects were presented there. The observed differences between species were fully expressed in isolated individuals. There was no evidence of the differences being magnified through interspecific competition. These differences, believed to be inherent, were considered sufficient to keep the species segregated without the involvement of competition. Segregation was concluded not to be of the interactive, type, even though the populations still retained considerable plasticity enabling them to switch diets or habitats when necessary or advantageous. The period of intense competition and food exploitation was considered to have occurred and ended during earlier stages of the coexistence. / Science, Faculty of / Zoology, Department of / Graduate

At a late stage of spermatogenesis a sperm-specific protein, protamine, is synthesized in the testis of salmonid fish and progressively replaces histones in combination with DNA. Protamine has a molecular weight near 5,000 and contains 2/3 of its total amino acid residues as arginine. Studies on the biosynthesis of protamine have been made on the rainbow trout (Salmo gairdnerii) testis. Successive column chromatography on Bio-Gel P-10 and CM- cellulose has been employed to isolate and characterize newly synthesized labelled protamine. Newly synthesized protamine is phosphorylated and is eluted earlier from the CM-cellulose column than mature protamine. However, the two forms of protamine chromatograph coin-cidentally when newly synthesized protamine is first treated with alkaline phosphatase. Protamine is separated into three components on CM-cellulose and the amino acid compositions of the components are very similar. The relative amounts of the components present in the testis nuclei are different at different stages of spermatogenesis and the synthesis of each component appears to be independently controlled. This suggests that the components, while chemically very similar, are the products of separate structural genes and may have different functions. By pulse labelling testis cell suspensions for different lengths of time and analyzing the amount of ¹⁴C- protamine found in the cytoplasm and in the nucleus, the site of protamine synthesis can be shown to be in the cytoplasm. Further, a cell-free, isolated post-mitochondrial cytoplasmic fraction can incorporate ¹⁴C-arginine into whole protamine molecules, while both an isolated nuclear fraction and high-speed supernatant were relatively inactive. This indicates that protamine synthesis occurs on cytoplasmic microsomes. Sedimentation analysis of pulse-labelled testis ribososes indicates that protamine is synthesized on a class of small polysomes, the disomes, sedimenting at 120S. While dimeric ribosomes investigated in various tissues have been shown to be inactive artefacts formed during isolation, the disomes in trout testis have been demonstrated to be a functional class of polysomes. They are not dissociable at 1 mM Mg⁺⁺ ion concentration, are not the breakdown product of larger polysomes, nor are they produced by interaction with free protamine. These disomes contain the major quantity of nascent protamine and increase in number in the testis cells during the active protamine synthesizing stage of development. The probable function of protamine is for the packaging of DNA into the sperm head. The phosphorylation of protamine and the protamine components may serve to regulate this packaging process. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Acute ammonia toxicity in rainbow trout was studied. This was carried out by injecting fish with various concentrations of ammonia dissolved in isotonic saline. In order to approximate conditions of natural toxicity, where ammonia would enter the fish at the gills, without the additional problems of environmental factors, the ammonium solutions were injected via a cannula implanted in the dorsal aorta. To determine if a differential toxicity existed in fish in relation to high pH and low pH ammonium solutions, ammonium bicarbonate and ammonium chloride were chosen for injection. Hydrogen ion, and total ammonia, concentrations were measured in blood sampled from the dorsal aorta, both before and after injection. In order to determine if, during the course of the injection, normal excretion rates would remove all of the injected ammonia, total ammonia and hydrogen ion concentrations were measured in blood sampled from both the dorsal and ventral aortae, and rates of extraction of ammonia from blood at the gills were calculated. Ammonia is toxic to fish. There was no significant difference between the dose of NH₄Cl and NH₄HCO₃ which killed fish. Therefore, unlike mammals, fish exhibited no differential toxicity to the ammonium compounds tested. Injection of NH₄Cl decreased pHa and injection of NH₄HCO₃ increased pHa. Both compounds increased the total ammonia concentration in the blood. Although in water the fraction of ammonia which exerts the toxic effects is unionized ammonia, within the fish it is the ionized fraction which exerts the toxic effects. The same dose of ammonium killed fish, but NH₄HCO₃-injected fish which survived had a much higher concentration of unionized ammonia in the blood than NH₄Cl-injected fish which died. Ammonia extracted from the blood in control fish was about one-fifth the amount which killed fish. This, together with the measured increases in blood ammonia following injection, demonstrate that, although ammonia is a normal excretory product of rainbow trout, the trout cannot increase excretion rates sufficiently to rid themselves of an ammonia load. Symptoms observed in fish following injection of ammonium solutions led to the conclusion that ammonia acts at the neural level. / Science, Faculty of / Zoology, Department of / Graduate

In this work, rainbow trout were used as a model system to examine the regulation of insulin (INS)- and insulin receptor (IR)-encoding mRNA expression profiles. INS- and IR-encoding mRNAs were isolated, cloned, and sequenced; and shown to be differentially expressed within and among multiple tissue types. Regulation was examined through various nutritional and hormonal treatments (in vivo and in vitro). A real-time quantitative-PCR assay was developed to measure the respective levels of mRNA expression. Fasting, growth hormone (GH), and somatostatin (SS) differentially regulated INS and IR mRNAs within selected tissues, in vivo. Glucose, GH, SS, and insulin-like growth factor-1 (IGF-I) differentially regulated INS and IR mRNAs within selected tissues, in vitro. The results of this dissertation research display the identification and differential regulation of multiple INS- and IR-encoding mRNAs and suggest that independent mechanisms may serve to regulate the various isoforms in a tissue-specific manner. Future studies are also suggested.