The foundation of the research compilation presented here began with the derivation of an improved method for obtaining optimal in vitro mitogenic responses in salmonid lymphocyte cultures by utilizing autologous or homologous plasma as the primary serum supplement. It was observed that lymphocytes which were previously unresponsive to mitogenic challenge in vitro, when cultured in fetal bovine serum, responded well when cultured in the presence of homologous plasma. Salmonid plasma sources not only repeatedly enhanced the mitogen-specific proliferation of the lymphocyte cultures, but enhanced the antibody response as well. A prolonged kinetic response further supported the contention that former conditions of salmonid lymphocyte culture, employing only fetal bovine serum, not only fail to provide the optimal conditions for cell growth, but in many cases the essential conditions. Enhancement of the mitogenic response was observed for three distinct species, using a common plasma source, suggesting utilization of plasma as an alternate serum supplement has broad applications and may be adapted to many fish systems. With this improved culture system, examination of the regulation of lymphocytes, specifically B cells, was undertaken. Evidence for the existence of a natural regulatory cell population located in the anterior kidney is presented. Addition of anterior kidney cells to either autologous peripheral blood or spleen cell cultures resulted in significant suppression of the mitogen response. The degree of suppressor activity appears to be correlated with the anterior kidney lymphocyte's ability to respond to mitogenic stimulus. It is demonstrated that a decrease in the anterior kidney mitogen response correlates significantly with an increase in the suppressor activity observed upon coculture of these same cells with peripheral blood lymphocytes. Interestingly, while addition of anterior kidney cells to spleen plaque forming cell cultures also resulted in suppression, anterior kidney cells had either no effect or enhanced the antibody response of the peripheral blood lymphocyte cultures. It is postulated that the mediator of this anterior kidney activity is a suppressor cell population which may possess an important immunoregulatory function. / Graduation date: 1992

Over recent years the aquaculture industry has grown at a faster rate than any other major animal food production sector. This growth in the size of the sector has created a need for research into the fundamental biology of the animals being farmed. This PhD examined the relationship between the immune response and growth/development using several different model systems. Chapter 3 investigated effects of an acute pro-inflammatory stimulation on the transcriptomic output of Atlantic salmon primary muscle cells in vitro. In response to immune stimulation with the pro-inflammatory cytokine IL-1β there was an increase in the expression of several pathways including: the acute phase response, muscle catabolism, growth and cell cycle regulation. These results provide some evidence that the molecular basis of immune related growth reduction may be the same in fish as in higher vertebrates. Chapter 4 investigated the effect of the parr/smolt transformation (PST), a developmental stage during which salmon exhibit reduced disease resistance, on the expression of immune related genes in response to acute immune stimulation. This involved the use of the bacterial mimic (LPS) and the viral mimic (poly I:C), to elicit an immune response from salmon at different stages over the PST. The magnitude of response to immune stimulation was suppressed in multiple tissues over the PST. Chapter 5 investigated the ability of Rainbow trout fry at both first feeding and three weeks post first feeding to respond to immune challenge. The immune response increased in magnitude and complexity with age. The effects of growth rate on immune gene expression were investigated; there was a slight increase in the up-regulation of expression in immune related genes and protein degradation genes in the slow growing fish. The findings in this thesis provide further understanding of the interaction between growth/development and the immune response.

Thesis (M.S.)--Humboldt State University, 2007. / Includes bibliographical references (leaves 32-37). Also available via Humboldt Digital Scholar.

There are currently thirteen members of the gonadotropin-releasing hormone (GnRH) family. The GnRH members that activate the synthesis and release of the pituitary gonadotropins are the best understood. These members stand central to the development and maintenance of reproductive function. The roles of GnRH that act in the brain and not in the pituitary, or that are expressed in extraneural tissues, are not well characterized. My goal was to determine whether 1) the regulatory regions and organization of the GnRH gene is conserved between mammals and fish, 2) GnRH is expressed in tissues other than the brain of salmonids and 3) the processes that regulate the expression of GnRH are conserved between two salmonid species with different habitats and reproductive patterns (sockeye salmon, Oncorynchus nerka; rainbow trout, Oncorynchus mykiss). To determine whether the regulatory regions and organization of the GnRH gene were conserved across the species, I isolated and characterized salmon (s)GnRH gene 1 from rainbow trout and sGnRH gene2 from sockeye salmon. In salmon, which are tetraploid, each duplicated sGnRH gene encoded a different mRNA (mRNA1 or mRNA2), but the identical sGnRH peptide. A Southern blot analysis revealed that other related forms of GnRH exist in the sockeye salmon genome. Also, I determined from RT-PCR analysis that GnRH was not expressed in the heart, liver, gut, adrenal, spleen or retina, but was expressed in the gonads of sockeye salmon. To understand the function of GnRH in the gonads, it was necessary to learn when GnRH was expressed during development and throughout the reproductive cycle. Studies using RT-PCR analysis and primer extension analysis demonstrated that the reproductive tissues of salmonids use an upstream promoter to regulate GnRH expression. Intron 1 may be retained, resulting in mRNAs containing 5'-untranslated regions longer than their brain counterparts. These sGnRH transcripts are initiated by a TATA-less promoter region from a start site at 315 basepairs upstream from that utilized in the sockeye salmon brain. Using the same techniques, differences in the expression of GnRH in embryonic tissue and gonads of sockeye salmon and rainbow trout were noted over the first two years of their lives. First, the upstream promoter is transiently used for expression of GnRH as early as 14 days after fertilization in rainbow trout and 30 days after fertilization in sockeye salmon. Second, in sockeye salmon ovary and testis, GnRH was expressed in October of the first year and then only during May and June of the second year in precocious tissue. For rainbow trout, GnRH was expressed in the first year from May to October and in the second year only in December. Precociously mature ovary and testis expressed GnRH from June to October in the second year. It was also important to determine whether the GnRH mRNA expressed in the developing ovary and testis was translated into protein. High pressure liquid chromatography and radioimmunoassays were used to demonstrate the presence of at least three forms of GnRH in precociously mature ovaries and testes during the second year. The expression of sGnRH mRNA2 in the salmonid ovary and testis utilizes an alternative promoter. The resulting mRNAs have long 5'- untranslated regions that may be important in post-transcriptional control. Expression of GnRH in the brain is constant, but is intermittant in the salmonid gonad. GnRH mRNA is expressed in undifferentiated gonadal tissue in the first year and briefly in differentiated, but immature gonads. However, in precociously mature ovary and testis in the second year GnRH is transcribed and translated at the stage that precedes ovulation and spawning. Differences in pattern and longer duration of GnRH expression are shown in the ovary and testis of rainbow trout in comparison to sockeye salmon. This might indicate that GnRH is important in the regeneration of new sets of germ cells in the iteroparous rainbow trout, but not in the semelparous sockeye salmon. A comparison of the genes that encode sGnRH mRNA1 and mRNA2 reveals significant sequence divergence in their 5’-flanking regions following tetraploidization. A large portion of the sockeye salmon gene2 is missing in comparison to the Atlantic salmon gene2. However, the salmonid genes all share strong sequence identity in the proximalprom oter region. Although large segments of sequence identity do not exist in the regulatory regions of the GnRH-encoding genes of mammals and salmonids, some similarities exist in the positions of potential POU- homeodomain regulator and estrogen response element motifs. This suggests that some regulatory control for expression of GnRH in both the brain and gonads may be conserved. / Graduate

Juvenile fish belonging to three genera and ten species of salmonids, were compared in field and laboratory studies. The main objectives were to describe the aggressive social behavior in a comparative way and to study those components of behavior associated with stream residence. In comparisons of aggregating tendencies of lake trout, rainbow trout, cutthroat trout, coho salmon and chum salmon it was found that chum fry formed relatively tight schools while the others did not. Coho fry aggregated when feeding and dispersed afterwards. Both rainbow and cutthroat showed a preference for shadowed portions of their containers. Rainbow selected the deep end of a tank graded from shallow to deep, while chum fry swam back and forth. Rainbow showed no selection for a small waterfall. It was shown that food, predatory fish and dominant individuals affected the selection of a location. From these experiments it was reasoned that resident and migrant fish have different innate environmental preferences but that these can be modified by external factors. The rate of locomotion was shown to be highest in migrant chum fry, lowest in resident fishes and intermediate in lake trout. The continuous swimming of chum fry was contrasted with the discontinuous swimming of residents. Again lake trout were intermediate. Standard observations were made of intraspecific groups in the laboratory. Resident fishes were aggressive and developed social hierarchies based on fighting, displays and nipping. Most nipping was performed by dominant fish and all of the activities of the subordinates were influenced by the presence of the dominant individual in their group. Some migrant fishes were aggressive, others were not. Chum fry nipped and chased each other but, evidently because of their continuous swimming movements, did not develop stabilized dominance orders. Lake trout, sockeye fry and pink fry were not aggressive and did not develop social hierarchies. Dominant coho fry attacked intruders more vigorously than they did members of their own group and the intruders often died. It was observed that intruders showed a characteristic "escape behavior" which may have identified them and singled them out for attack. In field studies carried out during different seasons, it was observed that individual coho fry exchanged positions and moved about within a home range. They did not appear to restrict themselves to private territories. During winter floods the home range was vacated and the fish occupied pools along the stream edge. In spring pre-migrant smolts were in pools while newly emerged fry were in shallow margins. Predation by smolts appeared to affect the location of the fry. Interspecific combinations were observed in the laboratory. Rainbow trout were most aggressive. The aggressive activities of spawning adults were similar to those of the juveniles but the actual reproductive movements were unique to the adults. Agonistic behavior appeared to be primitive in lake trout with increasing specialization through dolly varden to brook trout. It was highly developed with specific variations in displays in brook trout, cutthroat trout, rainbow trout and coho salmon. The other species within the genus Oncorhynchus exhibited a degeneration in residential behavior. / Science, Faculty of / Zoology, Department of / Graduate

The literature on Ichthyobodo necator is reviewed. The prevalence and intensity of Ichthyobodo infestations on farmed salmonids was investigated on three farms over a period of two years. The infestations were found to be markedly age dependent. Peak infestations and related mortalities occurred in the first eight weeks after first feeding. Both mortalities and infestations declined to zero shortly after this period with no chemotherapy. Ichthyobodo reappeared on 0+ and appeared for the first time on I+ fish after a drop of water temperatures to less than 10*C. Many of the 1+ fish had started to mature. It is suggested that some form of host defence mechanism operates which limits the Ichthyobodo infestations in farmed salmonids. The sequential pathology of Ichthyobodo infestations of the skin of 0+ and 1+ salmon and rainbow trout was studied. Areas of greatest shelter from water currents were found to be most commonly infested and no parasites were found attached to the epidermis on the head of the fish. The parasite caused hyperplasia of the malphigian cells and exhaustion of the goblet cells below infestations, followed by spongiosis of the underlying epidermis. The epidermal plaque then sloughed off leaving a single layer of cells attached to the basement membrane. Cell kinetic studies showed that Ichthyobodo caused the cells immediately below infestations to divide, a markedly different pattern from that of normal teleost epidermal cell proliferation. The possibility that the parasite secretes some form of digestive enzyme is postulated. In areas where sloughing had occurred, the remaining malphigian cells were seen to be in the process of division. Various endocrinological aspects of Ichthyobodo infestations were investigated. Three corticosteroids and one androgen were injected or implanted into 1 year old rainbow trout. Implantations of hydrocortisone led to very heavy ichthyobodo infestations. Radio immune assays showed that the level of cortisol and testosterone in the serum of implanted fish was similar to that which would occur when salmonids mature. There appears to be a clear link between cortisol levels in the serum and Ichthyobodo infestation. The host response to Ichthyobodo is discussed and it is concluded that cortisol may suppress the host's defence mechanism to Ichthyobodo.

No description available.

Little is known about the virulence factors of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. The predominant protein produced by R. salmoninarum in broth culture or during infection is a 57/58 kDa protein (p57) which is associated with strain virulence. In this study monoclonal antibodies (MAbs) to p57 were developed and used as tools to antigenically characterize and quantify the protein. Monoclonal antibodies 4D3 and 2G5 recognize p57 and appear to be species specific as they did not cross-react with proteins produced by bacterial species within the genera Streptococcus, Carnobacterium, Vibrio and Aeromonas, or with fish serum proteins. Further, these MAbs recognize conserved epitopes on p57 shared by 10 isolates from geographically diverse areas. In vitro activities attributed to p57 include the suppression of antibody production, and the agglutination of rabbit erythrocytes and salmonid spermatocytes. We described a novel in vitro agglutinating activity of p57 toward salmonid leukocytes that was inhibited by two of a panel of eight MAbs. The location of the putative epitopes recognized by the MAbs were determined by two-dimensional electrophoresis and Western blotting of proteolytic breakdown fragments of p57. Amino acid sequencing of several of the fragments suggested that the antibodies which inhibit agglutinating activity bind proximal to the amino terminus of the protein. To investigate the mechanism of leukocyte agglutination, p57 was purified to near homogeneity using anion-exchange and size-exclusion fastpressure liquid chromatography. P57 eluted as a protein monomer and retained leukoagglutinating activity. In addition, results of antibody-capture, enzyme-linked immunosorbent assays suggest that a monomer exists in culture supernatant and infected fish tissue. Antigenic analysis with MAbs has also been useful for developing immunoassays for detecting and quantifying p57 levels in vivo. Using a quantitative ELISA, the prevalence of salmon with antigen levels above 3 ng/ml of kidney homogenate varied from 12.8 to 36.6% in 740 adult spawning chinook salmon returning to an Oregon hatchery from 1989 to 1991. A rapid, semi-quantitative, Field ELISA was also developed for use under hatchery conditions, in addition to a sensitive chemiluminescent Western blot protocol for confirming ELISA positive samples. / Graduation date: 1992

Thesis (Ph. D.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

Thesis (M.S.)--University of Massachusetts Amherst, 2008. / Includes bibliographical references (p. 97-100).