Stress up-regulates oxidative burst in juvenile Chinook salmon leukocytes.

When fish perceive stressful scenarios, their hypothalamus-pituitary-interrenal axis is activated resulting in the release of corticotropin releasing hormone, adrenocorticotropic hormone (ACTH), and finally cortisol. The physiologic stress response of fish has most often been linked to the reduced performance of the immune system, with a few exceptions where the immune system is activated. In this report, we tested the hypothesis that oxidative burst activity levels in juvenile Chinook salmon (Oncorhynchus tshawytscha) are altered when the fish is presented with a stressor. Fish were subjected to a stressor for 3 h and then allowed to recover for 20 h following the stressor. Plasma and spleens were collected from euthanized fish before the stressor, at the end of a 3 h stressor, and 23 h after the start of the experiment. Plasma was held at -80 °C until cortisol radioimmunoassay analysis was performed to confirm stress. Spleens were held in Dulbecco's Modified Eagle Medium overnight and analyzed the day following collection. Oxidative burst activity was measured in splenic leukocytes after being stimulated with phorbol 12-myristate 13-acetate. We found a significant increase in activated oxidative burst from fish subjected to the stressor as compared to unstressed fish. Speculation is given to ACTH being the leukocyte priming agent in this experiment rather than the cortisol itself.

Swimming Endurance in Juvenile Chinook Salmon Infected with Salmincola californiensis.

Juvenile Chinook Salmon Oncorhynchus tshawytscha moving downstream through tributaries of the upper Willamette River basin can spend months in reservoirs created by dams. While residing in the reservoirs, they often obtain heavy infections of the freshwater parasitic copepod Salmincola californiensis. The physiologic effect these parasites have on salmonids is poorly understood. We developed a method to infect juvenile Chinook Salmon in a laboratory with the copepodid stage of S. californiensis. Infected and uninfected fish were subjected to a swimming challenge to ascertain swimming endurance. Severity of gill damage was assessed using a dissecting microscope. Juvenile Chinook Salmon naturally infected with S. californiensis in Cougar Reservoir, Oregon, were also challenged and compared with their lab-infected counterparts. Copepod infection greatly impaired the swimming ability of laboratory fish, and the naturally infected fish were entirely incapable of swimming at low velocity. Chinook Salmon collected in the wild were more heavily infected than the laboratory fish and had trouble surviving collection and transport to our laboratory. The intensity of infection and severity of gill damage were positively correlated with diminished swimming ability, suggesting that heavy infection with copepods impairs gas exchange and osmotic regulation, which likely results in diminished fitness and decreased survival of infected fish.

Growth and annual survival estimates to examine the ecology of larval lamprey and the implications of ageing error in fitting models.

This study used existing western brook lamprey Lampetra richardsoni age information to fit three different growth models (i.e. von Bertalanffy, Gompertz and logistic) with and without error in age estimates. Among these growth models, there was greater support for the logistic and Gompertz models than the von Bertalanffy model, regardless of ageing error assumptions. The von Bertalanffy model, however, appeared to fit the data well enough to permit survival estimates; using length-based estimators, annual survival varied between 0·64 (95% credibility interval: 0·44-0·79) and 0·81 (0·79-0·83) depending on ageing and growth process error structure. These estimates are applicable to conservation and management of L. richardsoni and other western lampreys (e.g. Pacific lamprey Entosphenus tridentatus) and can potentially be used in the development of life-cycle models for these species. These results also suggest that estimators derived from von Bertalanffy growth models should be interpreted with caution if there is high uncertainty in age estimates.

Body morphology differs in wild juvenile Chinook salmon Oncorhynchus tshawytscha that express different migratory phenotypes in the Willamette River, Oregon, U.S.A.

Body morphology of juvenile Chinook salmon Oncorhynchus tshawytscha in the upper Willamette River, Oregon, U.S.A., was analysed to determine if variation in body shape is correlated with migratory life-history tactics followed by juveniles. Body shape was compared between migrating juveniles that expressed different life-history tactics, i.e. autumn migrants and yearling smolts, and among parr sampled at three sites along a longitudinal river gradient. In the upper Willamette River, the expression of life-history tactics is associated with where juveniles rear in the basin with fish rearing in downstream locations generally completing ocean ward migrations earlier in life than fish rearing in upstream locations. The morphological differences that were apparent between autumn migrants and yearling smolts were similar to differences between parr rearing in downstream and upstream reaches, indicating that body morphology is correlated with life-history tactics. Autumn migrants and parr from downstream sampling sites had deeper bodies, shorter heads and deeper caudal peduncles compared with yearling smolts and parr from the upstream sampling site. This study did not distinguish between genetic and environmental effects on morphology; however, the results suggest that downstream movement of juveniles soon after emergence is associated with differentiation in morphology and with the expression of life-history variation.

Post-mortem sporulation of Ceratomyxa shasta (Myxozoa) after death in adult Chinook salmon.

Ceratomyxa shasta (Myxozoa) is a common gastrointestinal pathogen of salmonid fishes in the Pacific Northwest of the United States. We have been investigating this parasite in adult Chinook salmon ( Oncorhynchus tshawytscha ) in the Willamette River, Oregon. In prior work, we observed differences in the pattern of development of C. shasta in adult salmon compared to juvenile salmon. Adult salmon consistently had large numbers of prespore stages in many of the fish that survived to spawn in the fall. However, myxospores were rarely observed, even though they were exposed and presumably infected for months before spawning. We evaluated the ability of C. shasta to sporulate following fish death because it is reported that myxosores are common in carcasses of Chinook salmon. We collected the intestine from 30 adult salmon immediately after artificial spawning and death (T0). A total of 23 fish were infected with C. shasta based on histology, but only a few myxospores were observed in 1 fish by histology. Intestines of these fish were examined at T0 and T7 (latter held at 17 C for 7 days) using quantified wet mount preparations. An increase in myxospore concentrations was seen in 39% of these fish, ranging between a 1.5- to a 14.5-fold increase. The most heavily infected fish exhibited a 4.6-fold increase from 27,841 to 129,352 myxospores/cm. This indicates, supported by various statistical analyses, that under certain conditions presporogonic forms are viable and continue to sporulate after death in adult salmon. Considering the life cycle of C. shasta and anadromous salmon, the parasite may have evolved 2, non-mutually exclusive developmental strategies. In young fish (parr and smolts), the parasite sporulates shortly after infection and is released into freshwater from either live or dead fish before their migration to seawater, where the alternate host is absent. The second strategy occurs in adult salmon, particularly spring Chinook salmon, which become infected upon their return to freshwater in the spring or early summer. For several months throughout the summer, only prespore stages are observed in most fish, even at the time of spawning. But once the fish dies, environmental conditions experienced by C. shasta change and viable presporogonic stages are induced to sporulate. As the post-spawned fish occur in the upper reaches of rivers, the myxospores would be released in a freshwater environment that would provide a reasonable opportunity for them to encounter their freshwater polychaete hosts, which reside downstream.

Identification of biomarkers indicative of barotrauma and recovery in black rockfish Sebastes melanops.

A Sebastes-specific complementary DNA (cDNA) microarray was developed to identify potential biomarkers involved in the capture stress and recovery of Sebastes species if they are assisted in returning to their original depth of capture following barotrauma. Black rockfish Sebastes melanops were exposed to simulated decompression from 450 kPa (c. 35 m depth) (which resulted in barotrauma) and subsequent recompression. Sebastes melanops were sampled for liver tissue at days 3, 15 and 31 post-barotrauma. Potential candidate genes were identified from the microarray and then quantitative real-time PCR (qrt-PCR) was used to validate expression levels in biological replicates. Six potential biomarkers associated with the innate immune system were identified that were up-regulated in liver tissue at 3 days post-barotrauma: complement C1q-like protein 2, complement component C3, complement regulatory plasma protein, serum amyloid A-5, c-type lysozyme and hepcidin precursor type I. In addition, complement c1q was correlated to the presence of a ruptured swimbladder, providing further support that this gene may be a good biomarker of injury and recovery. Immune genes were no longer up-regulated at day 31 post-barotrauma, a good indication of recovery in S. melanops.

Recovery potential of black rockfish, Sebastes melanops Girard, recompressed following barotrauma.

Overfished species of rockfish, Sebastes spp., from the Northeast Pacific experience high bycatch mortality because of 'barotrauma', a condition induced from the rapid change in pressure during capture. Field experiments show that it may be possible for rockfish to recover from barotrauma if quickly recompressed; however, no work has followed the physiological recovery of rockfish after recompression or determined whether it is possible for rockfish to survive barotrauma in the long term. Barotrauma was induced in adult black rockfish, Sebastes melanops Girard, from a simulated depth of 35 m, followed by recompression. Blood and selected tissues (eye, heart ventricle, head kidney, liver, rete mirabile and gonad) were sampled at days 3, 15 and 31 post-recompression to evaluate the tissue- and physiologic-level response during recovery. No mortality from barotrauma occurred during the experiments, and feeding resumed in 80% of both treatment and control fish. The primary injury in treatment fish was the presence of a ruptured swimbladder and/or a ruptured tunica externa (outer layer of swimbladder), which was slow to heal. Blood plasma was analysed for glucose, sodium, chloride, potassium, calcium, phosphorus, insulin-like growth factor-1 and cortisol. Plasma analyses indicated no strong effects because of barotrauma, suggesting overall handling stress outweighed any effect from barotrauma. Rockfish with ruptured swimbladders may face compromised competency in the wild; however, it appears the majority of black rockfish decompressed from 35 m have a high potential for recovery if recompressed immediately after capture. This research suggests recompression could be a valuable bycatch mortality reduction tool for rockfish in recreational fisheries.

Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton).

Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities.

Using blood plasma for monitoring organochlorine contaminants in juvenile white sturgeon, Acipenser transmontanus, from the lower Columbia River.

Organochlorine (OC) pesticide concentrations in blood plasma samples from 88 juvenile white sturgeon collected from the lower Columbia River were measured and compared to plasma sex steroid and OC tissue levels previously measured in corresponding fish. Significant squared correlation coefficients between summation operator DDT concentrations in sturgeon plasma and gonads and livers were 0.37 and 0.32, respectively. Significant negative correlations between plasma testosterone concentration and plasma Sigma DDT concentration in male fish (r(2)=0.26), plasma 17beta estradiol concentration and plasma Sigma DDT concentration in female fish (r(2)=0.38) and condition factor and plasma Sigma DDT concentration in all fish were found (r(2)=0.17). These results suggest that blood plasma may be a suitable nondestructive method for monitoring adult sturgeon population for persistent OC contaminants.

Chronic administration of fluoxetine alters locomotor behavior, but does not potentiate the locomotor stimulating effects of CRH in juvenile Chinook salmon (Oncorhynchus tshawytscha).

The present study investigated: 1) the behavioral effects of chronic administration of a serotonin uptake inhibitor (fluoxetine) in juvenile Chinook salmon, Oncorhynchus tshawytscha and, 2) whether chronic administration of fluoxetine alters the behavioral effects of corticotropin-releasing hormone (CRH). Chronic (20 day) treatment with fluoxetine decreased locomotor activity when compared to fish given long-term injections of saline. An intracerebroventricular (i.c.v.) injection of CRH had no effect on locomotor activity following a 20 day intraperitoneal treatment with either saline or fluoxetine. Chronic treatment with fluoxetine also increased the amount of time fish spent near the center of the tank. A similar increase was seen in fish given a chronic intraperitoneal (i.p.) series of saline followed by an acute i.c.v. injection of CRH. However, the effect was not additive when fish were given chronic i.p. injections of fluoxetine followed by an acute i.c.v. injection of CRH. These results provide evidence to support the hypothesis that the serotonergic system is involved in mediating locomotor activity and habitat choice in teleosts.

Gene expression in the liver of rainbow trout, Oncorhynchus mykiss, during the stress response.

To better appreciate the mechanisms underlying the physiology of the stress response, an oligonucleotide microarray and real-time RT-PCR (QRT-PCR) were used to study gene expression in the livers of rainbow trout (Oncorhynchus mykiss). For increased confidence in the discovery of candidate genes responding to stress, we conducted two separate experiments using fish from different year classes. In both experiments, fish exposed to a 3 h stressor were compared to control (unstressed) fish. In the second experiment some additional fish were exposed to only 0.5 h of stress and others were sampled 21 h after experiencing a 3 h stressor. This 21 h post-stress treatment was a means to study gene expression during recovery from stress. The genes we report as differentially expressed are those that responded similarly in both experiments, suggesting that they are robust indicators of stress. Those genes are a major histocompatibility complex class 1 molecule (MHC1), JunB, glucose 6-phosphatase (G6Pase), and nuclear protein 1 (Nupr1). Interestingly, Nupr1 gene expression was still elevated 21 h after stress, which indicates that recovery was incomplete at that time.

Mercury concentrations in gonad, liver, and muscle of white sturgeon Acipenser transmontanus in the lower Columbia River.

This study determined the partitioning of total mercury in liver, gonad, and cheek muscle of white sturgeon (Acipenser transmonatus) in the lower Columbia River. The relationship between tissue mercury concentrations and various physiologic parameters was assessed. White sturgeon were captured in commercial fisheries in the estuary and Bonneville, The Dalles, and John Day Reservoirs. Condition factor (CF), relative weight (Wr), and gonadosomatic index (GSI) were determined for each fish (n = 57). Gonadal tissue was examined histologically to determine sex and stage of maturity. Liver (n = 49), gonad (n = 49), and cheek muscle (n = 57) were analyzed for total mercury using cold-vapor atomic fluorescence spectrophotometry. Tissue protein concentrations were measured by ultraviolet-visible spectroscopy. Plasma was analyzed for testosterone (T), 11-ketotestosterone (KT), and 17ss-estradiol (E2) using radioimmunoassay. Mean tissue mercury concentrations were higher in muscle compared with liver and gonad at all sampling locations, except Bonneville Reservoir where mean liver mercury content was the highest tissue concentration observed in the study. Significant negative correlations between plasma androgens (T and KT) and muscle mercury content and plasma E2 and liver mercury content were found. A significant positive linear relationship between white sturgeon age and liver mercury concentrations was evident. Significant negative correlations between CF and relative weight and gonad and liver mercury content were found. In addition, immature male sturgeon with increased gonad mercury content had decreased GSIs. These results suggest that mercury, in the form of methylmercury, may have an effect on the reproductive potential of white sturgeon.

Plasma androgen correlation, EROD induction, reduced condition factor, and the occurrence of organochlorine pollutants in reproductively immature white sturgeon (Acipenser transmontanus) from the Columbia River, USA.

White sturgeon (Acipenser transmontanus) support an active fishery in the Columbia River, but there is poor reproductive success within the impounded sections. The poor reproductive success has been attributed to hydroelectric development; however, water pollution could be a significant factor. White sturgeon plasma, liver, and gonad samples were collected from four Columbia River locations and a California aquaculture facility. Total length and weight of the fish were measured, and plasma samples were analyzed for testosterone (T), 11-ketotestosterone (KT), 17 beta-estradiol (E2), and vitellogenin. Liver samples were analyzed for chlorinated pesticides and polychlorinated biphenyls, ethoxyresorufin-O-deethylase (EROD) activity and histopathology. Gonads were examined histologically to assess sexual maturity and characterize any lesions. Significant differences by location existed for p,p'-DDE, EROD activity, and condition factor. Plasma T was negatively correlated with p,p'-DDE in males and females, and plasma KT was negatively correlated in males. These data indicate that pollutants could be adversely affecting white sturgeon in the Columbia River basin.

Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures.

The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21 degrees C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures.

Effects of stress on the reproductive performance of rainbow trout (Oncorhynchus mykiss).

We investigated the effects of stress over the final stages of sexual maturation on the reproductive performance of female rainbow trout, Oncorhynchus mykiss. Stress was administered over the period of early vitellogenesis (1.5 mo), late vitellogenesis-final maturation (1.5 mo), or during both periods (3 mo). Each stress treatment and control was triplicated, with eight females in each replicate (n = 24 fish per treatment). The eggs and progeny of each female were kept separate, and observations were made for 4 mo after transfer to rearing tanks. Fish that experienced stress during final maturation and those that were under stress during the whole experiment ovulated on average 2 wk earlier than the control group. In contrast, fish stressed during the period of early vitellogenesis ovulated at the same time as controls. Absolute fecundity and fertilization were not significantly affected in any treatment group, but significant differences in relative fecundity were found. Stress applied early in vitellogenesis resulted in smaller eggs and swim-up fry. No significant differences were found in juvenile weight 8 wk after hatching. Furthermore, we found no differences in survival of the progeny or resistance to the fish pathogen Vibrio anguillarum. Thus, mild acute stresses applied to rainbow trout females may affect certain reproductive performance parameters such as timing of ovulation and relative fecundity; however, the progeny of such stressed females perform as well as controls with regard to juvenile growth and disease resistance.

Physiological levels of testosterone kill salmonid leukocytes in vitro.

Adult spring chinook salmon (Oncorhynchus tshawytscha) elaborate high plasma concentrations of testosterone during sexual maturation, and these levels of testosterone have been shown to reduce the salmonid immune response in vitro. Our search for the mechanism of testosterone's immunosuppressive action has led to the characterization of an androgen receptor in salmonid leukocytes. In the present study we examined the specific effects that testosterone had on salmonid leukocytes. Direct counts of viable leukocytes after incubation with and without physiological levels of testosterone demonstrate a significant loss of leukocytes in cultures exposed to testosterone. At least 5 days of contact with testosterone was required to produce significant immunosuppression and addition of a "conditioned media" (supernatant from proliferating lymphocytes not exposed to testosterone) did not reverse the immunosuppressive effects of testosterone. These data lead us to conclude that testosterone may exert its immunosuppressive effects by direct action on salmonid leukocytes, through the androgen receptor described, and that this action leads to the death of a significant number of these leukocytes.

Endogenous steroid metabolism is indicated by fluctuations of endogenous steroid and steroid glucuronide levels in early development of the steelhead trout (Oncorhynchus mykiss).

Concentrations of endogenous steroids and their glucuronide conjugates fluctuated during early development in steelhead trout Oncorhynchus mykiss. Whole body content of sex steroids and steroid glucuronides of both bisexual and gynogenetic (all female) steelhead trout were quantified by radioimmunoassay. Concentrations of 17beta-estradiol (E2) and cortisol increased 2-4 days before hatch. Two days after hatch, 11-ketotestosterone (11KT) increased in concentrations in both gynogenetic and bisexual populations, and 11KT glucuronide concentrations increased in the gynogenetic population. Testosterone (T) and E2 concentrations were at their lowest at 39 days postfertilization (dpf) for T and 39 and 61 dpf for E2. Changes in levels of steroid glucuronides were not consistently parallel to free steroids through time. T-, E2-, and 17alpha, 20beta dihydroxyprogesterone glucuronides declined slower than their free forms. Based on fluctuating concentrations of all steroid glucuronides, both populations of fish demonstrated an ability to form glucuronide conjugates of all steroids at the embryonic stage. The changes in levels of both free steroids and their glucuronides during early development of the trout indicate that steroid metabolism is active during development. This study also implicates steroid metabolism as an integral part of embryonic and postembryonic development.

Brain-pituitary-gonadal axis during early development and sexual differentiation in the rainbow trout, Oncorhynchus mykiss.

Profiles of testosterone, 11-ketotestosterone, androstenedione, and estradiol were determined by RIA, and immunocytochemical techniques were employed to identify gonadotropin (GTH) I and II and gonadotropin releasing hormone (GnRH) in monosex and mixed sex populations of rainbow trout from 1 to 126 days postfertilization (dpf). Steroid levels were relatively high at 1 dpf and declined until 25 dpf. At 30 and 48 dpf (hatching) steroid levels increased slightly before they fell by 78 dpf and remained relatively constant thereafter. Trends toward differences in steroid content between males and females became evident around the time gonadal differentiation was histologically discernible (78 and 90 dpf). GTH I was present in the proximal pars distalis at all dates (48-126 dpf), whereas GTH II was not detectable. GnRH was found at all dates (48-126 dpf) and was distributed in several areas of the brain including the nucleus preopticus periventricularis, nucleus lateralis tuberis, and the pituitary in the region where GTH I was found. No differences were seen between males and females in the timing of appearance, localization, or intensity of staining of these peptide hormones. Given that the brain-pituitary-gonadal axis seems to be intact during the process of sexual differentiation and the fluctuations of steroid levels during this process, sex steroids may play the driving role for sexual differentiation of rainbow trout.

In vivo steroid metabolism in embryonic and newly hatched steelhead trout (Oncorhynchus mykiss).

Radioactive pregnenolone (P5), testosterone (T), or 17-beta-estradiol (E2) was microinjected into steelhead trout, Oncorhynchus mykiss, embryos and newly hatched yolk-sac fry (alevins) to detect in vivo metabolism. We also assayed the water used to incubate animals for 10 hr after microinjection to detect possible metabolite excretion. High pressure liquid chromatography and thin layer chromatography were used to separate and tentatively identify steroid metabolites. Metabolites of P5 were androstenedione (AN), E2, T, and glucuronides of E2 and T in embryos and AN, E2, progesterone, 17-alpha, 20-beta-dihydroxyprogesterone, and P5 glucuronide in alevins. E2 and its glucuronide were synthesized from precursor T in the embryos and alevins; however, the amounts of E2 and E2 glucuronide synthesized in the embryos were 10 and 3 magnitudes greater than those detected in alevins. Testosterone glucuronide was synthesized in similar amounts in both stages of development. Embryos did not synthesize free metabolites from E2 precursor, but E2 glucuronide was detected from E2 precursor. Estradiol in alevins was metabolized into unidentified free and glucuronide-conjugated steroids. Three unknown metabolites synthesized from P5 precursors and seven unknown substances produced in animals injected with testosterone or estradiol precursors were detected. Free metabolites were detected in the incubation water that held the animals (embryos and alevins) for 10 hr after microinjection with T or E2. Glucuronide metabolites were not excreted by embryos into the incubation water 10 hr after microinjections with any of the steroid precursors; however, alevins excreted glucuronides into the incubation water when supplied with precursor T. These results imply that endogenous steroid metabolism of maternally contributed steroids is active during early development.