The long-toed salamander (Ambystoma macrodactylum) is the top vertebrate predator in fishless high-elevation lakes in North Cascades National Park Service Complex (NOCA), Washington. The purpose of this research was to determine the effects of physico-chemical factors and introduced trout on abundance and behavior of A. macrodactylum larvae. Although high-elevation lakes in NOCA were naturally fishless, trout were stocked in many lakes during this century to provide recreational angling opportunity. Twenty fishless lakes and 25 lakes with fish were sampled from 1990 to 1994. Larval salamander density and behavior were assessed by snorkeling lake perimeters and searching through nearshore substrate material and aquatic vegetation. In fishless lakes, larval salamander densities were positively related to total Kjeldahl-N concentration (TKN) and negatively related to lake elevation. Based on analysis of salamander stomach contents, crustacean zooplankton, especially cladocerans, were important food resources for larval A. macrodactylum. Total crustacean zooplankton, as well as cladoceran densities, were positively related to TKN concentration, suggesting that increased zooplankton food resources contributed to increased densities of A. macrodactylum. The effects of fish introductions on larval salamander densities depended on TKN concentration and whether or not trout had established reproducing populations. Mean larval salamander densities for fishless lakes with TKN<0.05 mg/l, generated from a linear regression equation with TKN and lake elevation as independent variables, were not significantly different from mean larval densities in lakes with either reproducing trout or in lakes with non-reproducing trout. However, in fishless lakes with TKN���0.05 mg/l, mean larval densities were significantly higher than in lakes with reproducing fish where trout reached high densities. In fishless lakes with TKN���0.095 mg/l, mean larval densities were significantly higher than in lakes with non-reproducing fish where trout fry were periodically stocked at low densities. Reduced larval salamander densities in lakes with trout likely was a consequence of trout predation. Although most larvae were associated with boulder, cobble, and woody debris substrates in nearshore areas of NOCA lakes, differences in the proportion of larvae hidden in benthic substrates between lakes with fish and without fish were not statistically significant. / Graduation date: 1997

Currently little is known about the mechanisms and locations of lymphocyte development in teleosts. In this study several aspects of the underlying factors which govern B lymphocyte development in trout were investigated which included: the isolation and characterization of immunoglobulin heavy chain (IgH) genes, the recombination activating genes 1 and 2 (RAG1 and RAG2) and the use of cellular markers to identify tissues harboring precursor B-cells. Immunoglobulin heavy chains are part of the structural components which make up antibody molecules produced by B-cells. We isolated various full-length IgH cDNA clones, some of which contained the secreted while others contained the membrane bound form of IgH. Upon characterization of the membrane bound forms, typical features common to all IgH cDNAs were found including a leader peptide, a variable region and constant domain containing transmembrane (TM) segments as well. Further sequence analysis of this region revealed that the TM domains were spliced directly to the CH3 domains which results in the loss of the entire CH4 region. Our results support previous observations of unusual splicing events in fish IgH genes. RAG1 and -2 in mammals have been shown to be essential for carrying out V (D) J recombination of lymphocyte receptors and are found to be expressed within primary lymphoid tissues and precursor lymphocytes. We isolated the RAG locus from a rainbow trout genomic library and characterized their conservation and expression. Overall the complete amino acid sequences of RAG1 and RAG2 displayed 78% and 75% similarity when compared to RAG genes from higher vertebrates thus demonstrating the highly conserved nature of these genes. Tissue specific expression of both genes was primarily associated with the thymus and pronephros in both juvenile and adult trout. Based upon these observation we conclude that the thymus and pronephros likely serve as the tissue sites for V (D) J recombination in trout and are thus primary lymphoid organs. Finally we addressed the question as to where B-cell lymphopoiesis occurs in trout. Our results using both immunofluorescence and confocal microscopy putatively demonstrate that the thymus harbors precursor B-cells and thus alludes to a dual function for both B and T-cell development in trout. / Graduation date: 1996

Graduation date: 1996

The relationship of coastal cutthroat trout (Oncorhynchus clarki clarki) populations in the Muck Creek basin, a 238 km�� southern Puget Sound stream basin in western Washington, was examined using starch gel electrophoresis and meristic analysis. Coastal cutthroat trout were collected from six sites throughout the basin including tributaries, portions of the mainstem, and a lake. Four sites contained only resident trout, the lower mainstem contained resident and anadromous trout, and the lake contained only mature anadromous trout based on size and appearance. Patterns of allelic and meristic variation suggest a significant structuring and separation of coastal cutthroat trout populations in the basin. The lake population was distinguished from the other populations by significant differences in allele frequencies and meristic characters. The other sites grouped more closely together with significant variation among and between sites meristically and at several loci. / Graduation date: 1996

Graduation date: 1995

Graduation date: 1995

Populationer av öring består av både migrerande och stationära individer, och det tycks vara både genetiska och miljömässiga faktorer som påverkar en individs beslut att vandra eller ej. Syftet med denna studie var att testa vilken effekt tillgång på föda har på tillväxt och smoltifieringsprocess hos öring (Salmo trutta). Min hypotes var att öring med hög tillförsel av mat växer bättre och smoltifieras därmed inte till samma grad som öring med låg tillförsel av mat. Som en konsekvens av skillnader i mattillgång förväntades att ATPas-aktiviteten, vilken indikerar smoltstatus, var lägre vid god tillförsel av mat än vid låg tillförsel. Experimentet utfördes från november 2003 till april 2004 vid fiskeodlingen i Brattfors, Värmland, där 2+ Klarälvsöringar utsattes för tre olika födonivåer, med fyra replikat av varje födonivå. Var 4-5 vecka fångades fisken för att vägas och mätas och slutligen togs biopsi på öringens gälar för en analys av Na+,K+ -ATPas aktivitet. I slutet av experimentet klassificerades fiskarna som parr eller smolt. Parr hade en signifikant lägre Na+, K+ -ATPas aktivitet än smolt i de tre olika födonivåerna. Na+, K+ -ATPas aktiviteten visade dock ingen signifikant skillnad mellan födonivåerna. Parr hade ökat sin vikt och längd mest i samtliga födonivåer och störst skillnad var det i den lägsta födonivån, parr hade ökat i genomsnitt 17 g och 3,5 mm mer än smolt. Av de två högsta födonivåerna var andelen klassificerade som smolt 37 % och vid den lägsta födonivån var andelen smolt 76 %. / Populations of brown trout consist of both migratory and resident individuals, and it is likely that both genetic and environmental factors influence the decision to migrate. The purpose of this study was to test the effect of food availability on growth and the smolting process in brown trout (Salmo trutta). My hypothesis was that brown trout subjected to a high food supply grows better and smoltifies to a lesser degree than brown trout subjected to a low food supply. As a consequence of differences in food supply I expected that ATPase-activity, which indicates smoltification status, would be lower with a high food supply than with a low supply. The experiment was run from November 2003 until April 2004 in a fish hatchery in Brattfors, Värmland, where 2+ brown trout from the river Klarälven were exposed to three food levels, each replicated four times. Every 4-5 weeks the fish were weighed and measured and a biopsy was taken from the gills for analysis of Na+, K+ -ATPase activity at the end of the experiment. At the end of the experiment the fish were classified as parr or smolt. Parr had a significantly lower Na+, K+ -ATPase activity than smolt for the three different food treatments. Na+, K+ -ATPase activity did not differ significantly between food levels. Parr grew faster (both weight and length) than smolt at all food levels, and the largest difference between parr and smolt was observed at the lowest food level, where parr increased on average 17 g and 3,5 mm more than smolt. At the two highest food levels the proportion classified as smolt was 37 % and at the lowest foodlevel the proportion of smolt was 76 %.

The organochlorine pesticide endosulfan has been shown to be highly toxic to fish and there is some evidence to support that it may act as an endocrine disrupting chemical. Juvenile rainbow trout (Oncorhynchus mykiss) were caged at 4 sites in Oshawa Creek during the fall and spring of 2008 and 2009 while another group was intra-peritoneal injected in the laboratory with varying concentrations (ppm) of endosulfan. Plasma vitellogenin (VTG) levels, liver ethoxyresorufin-O-deethylase (EROD), citrate synthase (CS), lactate dehydrogenase (LDH), and brain acetylcholine esterase (AChE) (caged fish only) enzymatic activities were measured. Trout injected with endosulfan experienced an increase of the anaerobic (LDH activity) and a decrease of the aerobic (CS activity) metabolic pathways, while male VTG levels increased. Since it was a singular injection, VTG results have to be confirmed. Fall caged trout showed increased EROD activity and inhibited AChE activity while those caged in the spring experienced an unexpected exposure to the lampricide 3-Trifluoro-Methyl-4-Nitro-Phenol (TFM) which disrupted metabolic parameters (inhibited CS and increased LDH activity). Both fall and spring caged trout experienced no induction of VTG activity. Further research is needed since the spring exposure was altered due to the unplanned TFM treatment and thus did not represent a valid temporal replicate.

Previous work (Thorgaard, G. H. et al., Aquatic Toxicology 46:121-126, 1999) showed triploid rainbow trout (0. mykiss) given embryonic carcinogen bath exposures had significant reduction of induced tumors relative to diploids. In the present study, trout were made triploid by thermal shock after fertilization. At age of 5 months they were given dietary carcinogen: aflatoxin B1 (AFB₁) or 7,12-dimethylbenz[a]anthracene (DMBA) for 30 or 120 days. The dietary exposures were at known tumorigenic levels (100, 200 and 300 ppb AFB₁; 250, 500 and 850 ppm DMBA). At about 16 months after fertilization the fish were sacrificed and tumor incidence and multiplicity were assessed. At all levels of carcinogen and in all tumorous organs tumor incidence was lower in the triploid fish. For DMBA-fed fish it was seen that the diploid:triploid incidence ratios ranged from 2.0 to 9.0 and for AFB₁ from 3.1 to 6.0. Weight class analyses dissociated the tumor incidence effects of growth from the effects of triploidy. Weight classes plotted against logit tumor incidence at all doses and durations showed parallel logistic lines. In every case the triploid curve was substantially lower than the diploid curve, showing the independent suppressive effect of triploidy. Fifteen triploid DMBA liver tumors were examined by direct cycle-sequencing of p53 PCR products across the exons 5, 7 and 8 known to contain nearly all human tumor p53 mutations. There were no p53 mutations seen at, or above, the present threshold of detection, (for radiolabeled manual sequencing, under 5% of mutant in normal). Fluorescent sequencing of 15 stomach tumors, also showed no p53 mutations in the hotspot-containing exons. Mutation detection by sequencing the trout Ki-ras1 gene, ortholog human KRAS2, showed codons 12, and 61 mutations in DMBA-fed trout liver and stomach tumors. The DMBA liver tumor Ki-ras1 mutation incidence showed no change by ploidy. There was a significant reduction in Ki-ras1 exon 1 mutations in triploid stomach tumors (5% in triploids v. 33% in diploids, Fisher's Exact test p<O.O5). AFB₁ liver tumors showed Ki-ras1 mutation incidence of 75% (9/12) in diploids and 90% (9/10) triploids, nearly all in exon 1, this mutation difference with respect to ploidy did not reach significance. / Graduation date: 2001

Previous studies have shown that rainbow trout exposed to dieldrin via diet for 9 to 12 weeks increased biliary excretion of a subsequent dose of [¹⁴C]dieldrin by 500%. This was not explained by induction of the cytochrome P-450 (CYP) system involved in oxidative metabolism of these compounds. We hypothesized that epoxide hydrolase activity increased in dieldrin fed-fish. Epoxide hydrolase is an enzyme that catalyzes the hydrolysis of epoxide compounds to their corresponding diols. For instance, dieldrin is metabolized to 6,7 trans-aldrindihydrodiol. This study investigated the activity of epoxide hydrolase in microsomes and cytosol of rainbow trout fed a diet that contained 0 or 15 ppm dieldrin. Fish were fed control or dieldrin diet (0.324 ug/g body weight/day) for 3, 6, or 9 weeks. There was a small increase in mortality and decrease in body weight among dieldrin-fed fish after 9 weeks. After week 9, dieldrin-fed fish were fed a control diet for an additional 3 weeks because of these signs of toxicity. At week 12, the difference of body weight between control and treated was not significant. Microsomal and cytosolic epoxide hydrolase activities were measured with a radiometric assay which determined differential partitioning of the parent compound (epoxide) in dodecane and the metabolite (diol) in the aqueous phase. Assays were run at optimal pH and temperature using [³H]trans-stilbene oxide (pH 7) as substrate for cytosol and [³H]cis-stilbene oxide (pH 8) as substrate for microsomes. In order to prevent competition for reaction with stilbene oxide, depletion of glutathione was efficiently achieved by dialysis at 4°C for 2 hours at room temperature in buffer [pH 7.5, potassium phosphate 10 mM, KCL 0.15 M, EDTA 1 mM, BHT 0.1 mM, 0.1 mM PMSF]. Protein quantification was determined by using BCA assay and concentrations were always between 5 and 25 ug/ml in the final assay volume. Epoxide hydrolase activities were not significantly different in cytosol or microsomes from control and dieldrin-fed fish. Dieldrin residues in liver were analyzed by gas chromatography with electron capture detection (GC/ECD). The concentration in the liver increased with time of exposure and declined markedly in fish fed dieldrin for 9 weeks and then fed control diet. No dieldrin was detected in livers from control fish. / Graduation date: 2003