Salmonids are a diverse group of fishes whose common ancestor experienced an evolutionarily important whole genome duplication (WGD) event approximately 90 MYA. This event has shaped the evolutionary trajectory of salmonids, and may have contributed to a proliferation of the repeated DNA sequences known as transposable elements (TEs). In this work I characterized repeated DNA in five salmonid genomes. I found that over half of the DNA within each of these genomes was derived from repeats, a value which is amongst the highest of all vertebrates. I investigated repeats of the most abundant TE superfamily, Tc1-Mariner, and found that large proliferative bursts of this element occurred shortly after the WGD and continued during salmonid speciation, where they have produced dramatic differences in TE content among extant salmonid lineages. This work provides important resources for future studies of salmonids, and advances the understanding of two important evolutionary forces: TEs and WGDs. / Graduate / 2019-04-19

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben. / Bodies of Water are both vital resources and pollutant sinks for a multitude of contaminants. Therefore, biological effect tests and biomonitoring of aquatic samples are of particular importance to detect potential environmental hazards. The "comet assay" (single cell gel electrophoresis) is an indicator for DNA strand breaks and was explored and applied as a genotoxicity test in the present study. Known genotoxic substances were used to determine the detection limits and dose-response relationships for the cell lines RTG-2 and RTL-W1 (from rainbow trout, Oncorhynchus mykiss) in vitro, and to adapt methodological parameters to the cells. The test was very sensitive to 4-Nitroquinoline-1-oxide. This substance was therefore well-suited to serve as positive control in further experiments. In order to evaluate the measurement data, an appropriate statistical procedure was developed, which also took "historical" controls into account. The time course of DNA damage in the test system using RTG-2 cells was determined, and relationships between the origin of DNA strand breaks, DNA repair capacity and the metabolizing capacity of the cells was investigated by means of inhibition of DNA repair with Aphidicoline. In the second stage, native water samples from the rivers Rhine and Elbe and further surface waters were tested with the comet assay, using RTG-2 cells. 15 out of 49 samples showed genotoxic effects. In a third stage, erythrocytes of feral chub, Leuciscus cephalus, from the Moselle river were examined with the comet assay. The fish from three measuring stations showed elevated values of DNA damage compared to fish sampled from a downstream station. There were only partly correlations with the results from additional biomarkers. Chemical analyses of water and tissue samples did not permit conclusions on effect-causing substances.However, this emphasizes the importance of biological tests in dealing with complex environmental samples.

Rainbow trout (Oncorhynchus mykiss) are introduced in large parts of the world and are commonly farmed for consumption as well as a valued sport fish. Many species of salmonids show large intraspecific variation in pigmentation, which has been shown to correlate with stress response and several other behavioral traits. In this study, the behavior and stress response of rainbow trout linked to pigmentation is investigated, depending on previous sport fishing experience. Through data from a previous study on rainbow trout in semi-natural ponds, locomotor activity was measured as a proxy of stress, under three different sport fishing treatments. From previous photos, the pigment spots of each rainbow trout are counted to test the correlation with locomotor activity. In addition, it is tested whether different previous experience of sport fishing means a higher stress response when re-exposed to fishing. No significant relationship between pigment spots and locomotor activity could be obtained for the three treatment groups. However, significant differences in locomotor activity due to angling experience between treatment groups upon re-exposure to fishing were found, with fish that were inexperienced in angling having higher locomotor activity compared to previously caught fish. Sport fishing and catch-and-release had the effect of reducing locomotor activity in rainbow trout, which can be interpreted as fishing could both stress them and trigger the fish's feeding response, depending on previous experience. Fishing is believed to create a passivation due to the negative association of being caught, while fish not previously caught were activated by sport fishing. The study's missing correlation between pigment spots and stress has been both confirmed and denied in other studies and may depend on the origin and the degree of domestication, which means that the correlation within other species of salmonids or fish with different origins would be relevant to investigate. / Regnbåge (Oncorhynchus mykiss) förekommer introducerade i stora delar av världen och är vanliga att odlas för konsumtion samt en uppskattad sportfisk. Många arter av salmonider visar stor intraspecifik variation i pigmentering, vilket visats korrelera med stressrespons och flera andra beteendemässiga karaktärsdrag. I denna studie undersöks regnbågarnas beteende och stressrespons kopplat till pigmentering, beroende på tidigare erfarenhet av sportfiske. Genom data från en tidigare studie på regnbåge i semi-naturliga dammar mäts rörelseaktivitet som indirektmått på stress, under tre olika sportfiskebehandlingar. Från tidigare foton räknas varje regnbåges pigmentfläckar för att testa korrelationen med rörelseaktivitet. Därtill testas om olika tidigare erfarenhet av sportfiske, innebär högre stressrespons vid återexponering för fiske. Inget signifikant samband mellan pigmentfläckar och rörelseaktivitet kunde erhållas för de tre behandlingsgrupperna. Signifikanta skillnader i rörelseaktivitet på grund av erfarenheten av sportfiske mellan behandlingsgrupperna vid återexponering för fiske fanns dock, där fisk som var oerfaren sportfiske hade högre rörelseaktivitet jämfört med fisk som fångats tidigare. Sportfiske och catch-and-release hade effekten att minska rörelseaktiviteten hos regnbågar, vilket kan tolkas som att fisket både kunde stressa och trigga fiskens födorespons, beroende på tidigare erfarenhet. Fisket tros skapa en passivisering på grund av den negativa associationen att bli fångad, medan fisk som inte fångats tidigare aktiverades av sportfisket. Studiens uteblivna samband mellan pigmentfläckar och stress har både bekräftats och dementerats i andra studier och kan bero på ursprung och graden av domesticering, vilket gör att sambandet inom andra arter av salmonider eller fisk med olika ursprung vore aktuellt att undersöka.

The evolution and maintenance of multiple phenotypes within populations is an interesting evolutionary and ecological question, and is becoming increasingly important for the restoration and conservation of partially migratory species. In particular, why individuals adopt a particular phenotype has been the focus of numerous research efforts. In the partially migratory species Oncorhynchys mykiss (commonly known as steelhead and rainbow trout), I found that sex plays a strong role in whether an individual adopts an anadromous or resident life history. I observed a higher frequency of females adopting the anadromous tactic throughout the species range, and I attributed this to the relationship between body size and reproductive success in females that is not present in males. I did not observe differences in female bias along gradients of latitude or migration distance, and I hypothesize that while the frequency of tactics among populations may change along these gradients, either the change is equal between males and females, or an interaction of latitude and migration distance may control changes. I did not observe a corresponding male bias in the one resident population I sampled, and suggest that this is the result of a high frequency of residents in that population. I propose that sex ratios could be a valuable metric for measuring frequency of life-history tactics in populations and could facilitate the investigation into the controls of life-history tactics across broad scales. / Graduation date: 2013