The relationship between emergence from spawning gravel and growth in farmed rainbow trout Oncorhynchus mykiss.

The relationship between the timing of emergence from spawning gravel and growth after emergence was investigated in farmed Oncorhynchus mykiss. A relationship between the time of emergence and growth became evident after 6 months of rearing, where individuals with an intermediate emergence time had grown larger compared with early and late emerging individuals.

Context-dependent responses to novelty in Rainbow trout (Oncorhynchus mykiss), selected for high and low post-stress cortisol responsiveness.

Previous studies in a rainbow trout model, selectively bred for high (HR) and low (LR) post stress plasma cortisol levels, have yielded data that are indicative of contrasting stress coping styles. Fish from the HR line have been suggested to display a more diverse behavioral repertoire in challenging situations than the LR counterpart. The present study addressed whether such variation in behavioral flexibility traits was evident in different experimental settings using these selection lines. The fish were subjected to three sets of challenges (novel object test, resident-intruder test and confinement stressor test), all which were repeated a week later. Introducing a novel object evoked a divergent behavioral response in association with feeding: fish from the LR line displayed consistently suppressed feed intake while the HR fish remained unaffected. This observation was found to be repeatable along with attack latency and movement activity from the resident-intruder and confinement stressor tests. These results indicate that the behavioral responses in this animal model are context-dependent and shed new light on the expression of behavioral flexibility.

Effects of maternal stress coping style on offspring characteristics in rainbow trout (Oncorhynchus mykiss).

Maternal size, age, and allostatic load influence offspring size, development, and survival. Some of these effects have been attributed to the release of glucocorticoids, and individual variation in these stress hormones is related to a number of traits. Correlated traits are often clustered and used to define the proactive and reactive stress coping styles. Although stress coping styles have been identified in a number of animal groups, little is known about the coupling between stress coping style and offspring characteristics. In the present study, plasma cortisol levels in ovulated mothers and cortisol levels in non-fertilized eggs from two rainbow trout (Oncorhynchus mykiss) strains selected for high (HR) and low (LR) post-stress plasma cortisol levels were compared. Offspring characteristics such as egg size, larval growth, and energy reserves also were compared between the two strains. Maternal plasma and egg cortisol levels were correlated, but no difference between the HR and LR strains was detected in either parameter. LR females produced larger eggs, and larvae with larger yolk sacs compared to HR females, however no differences in larval body size (excluding the yolk) was detected between strains. Considering that the HR and LR strains have a number of correlated behavioral and physiological traits that resemble the reactive and proactive stress coping styles, respectively, the results suggest that proactive mothers invest more energy into their offspring, producing larvae with larger energy reserves. It is possible that larger energy reserves in proactive larvae support the energy requirement for establishing and defending territory in salmonid fish. Furthermore, in the present study we found a positive relationship between mother plasma cortisol and egg cortisol; however neither mother plasma cortisol nor egg cortisol differed between strains. These results indicate that cortisol endowment from the mother to the offspring plays a minor role in the transfer of the behavioral and physiological traits which separates these strains.

Consistent boldness behaviour in early emerging fry of domesticated Atlantic salmon (Salmo salar): Decoupling of behavioural and physiological traits of the proactive stress coping style.

Individual variation in the way animals cope with stressors has been documented in a number of animal groups. In general, two distinct sets of behavioural and physiological responses to stress have been described: the proactive and the reactive coping styles. Some characteristics of stress coping style seem to be coupled to the time to emerge of fry from spawning redds in natural populations of salmonid fishes. In the present study, behavioural and physiological traits of stress coping styles were compared two and five months after emergence in farmed Atlantic salmon (Salmo salar), using individuals with an early or late time to emerge. Initially, compared to late emerging individuals, early emerging individuals showed a shorter time to resume feeding after transfer to rearing in isolation. Resumption of feeding after isolation was suggested to be related to boldness behaviour, rather than hunger, in the present study. This observation was repeated five months after emergence, demonstrating behavioural consistency over time in this trait. However, in other traits of proactive and reactive stress coping styles, such as social status, resting metabolism or post stress cortisol concentrations, early and late emerging individuals did not differ. Therefore, this study demonstrates that boldness in a novel environment is uncoupled from other traits of the proactive and reactive stress coping styles in farmed salmonids. It is possible that this decoupling is caused by the low competitive environment in which fish were reared. In natural populations of salmonids, however, the higher selection pressure at emergence could select for early emerging individuals with a proactive coping style.

Temporal patterns of limbic monoamine and plasma corticosterone response during social stress.

Dominant and subordinate males respond differently to the stress of social interaction. After an hour of social interaction, subordinate male Anolis carolinensis have elevated serotonergic activity in hippocampus, but dominant males do not. In other species, and using other stressors, the activation of hippocampal serotonergic activity is much more rapid than one hour. To elucidate early stress responsiveness, adult male A. carolinensis were divided into four groups: isolated controls, and pairs of males sampled after 10, 20 or 40 minutes of aggressive interaction. Development of dominant-subordinate relationships was determined by behavior and by the celerity of eyespot darkening. Serotonergic activity in the hippocampus, nucleus accumbens and amygdala was elevated rapidly and equally in both dominant and subordinate males, as were plasma corticosterone concentrations. Serotonergic activity remained elevated through 40 minutes in hippocampus and nucleus accumbens. Only subordinate males had elevated corticosterone levels at 40 minutes. Social status does not impede socially induced stress responses. Rather, rapid regulation of serotonergic stress responses appears to be a mediating factor in determining both behavioral output and social status. Temporal expressions of monoaminergic and endocrine stress responses are distinctive between males of dominant and subordinate social status. Such temporal patterns of transmitter and glucocorticoid activity may reflect neurocircuitry adaptations that result in behavior modified to fit social status.

Radiation quality dependence of DNA damage induction.

Analysis of DNA fragmentation and repair in relation to radiation quality may give important information about the role of break complexity and correlated double strand breaks (DSBs). DNA fragment analysis was performed by pulsed-field gel electrophoresis after exposure to different radiation qualities. Normal human fibroblasts were irradiated with boron ions (40, 80 and 160 keV.micron-1), nitrogen ions (80, 125, 175 and 225 keV.micron-1) and neon ions (225 and 300 keV.micron-1). The amount of DNA less than 1.1 Mbp decreased with increasing linear energy transfer (LET) for all three ions. When theoretical random distributions were subtracted from the experimental data for 225 keV.micron-1 nitrogen ions in all size intervals (5-5700 kbp), there was a significant non-random distribution of DSBs for sizes up to 1-3 Mbp. This non-random distribution of breaks, probably produced by intra-track correlated DSBs, may constitute a substantial portion of the high-LET induced DSBs.

Rejoining of double-stranded DNA-fragments studied in different size-intervals.

PURPOSE: To measure rejoining of radiation-induced doublestranded DNA-fragments of different sizes and to evaluate the effects of size-resolution in the analysis of rejoining. MATERIAL AND METHODS: Normal human fibroblasts (GM5758) were irradiated with photons or accelerated nitrogen ions (linear energy transfer, LET = 125 keV microm(-1)) and incubated for repair for 0-22 h. Double-stranded DNA-fragments from the irradiated cells were separated by pulsed-field gel electrophoresis in the range approximately 5 kbp to 5.7 Mbp. RESULTS: For cells irradiated with high LET nitrogen ions, there was an increase in the fast half-time from approximately 5 min for fragments < 400 kbp to 10 min when all fragments < 5.7 Mbp were measured. Further, the fraction of fragments rejoined by the slow-rejoining phase increased significantly for increased threshold sizes. The fraction of unrejoined fragments after 22 h and the half-time for the slow-rejoining phase remained constant for all threshold sizes. For cells irradiated with lower doses of low LET radiation the rejoining was shifted towards a slower kinetics when fragments up to 10 Mbp were excluded in the analysis. CONCLUSION: DNA exclusion-size and resolution may affect the estimates of DNA double-strand break rejoining. Using a low-resolution technique that does not detect small fragments will result in an underestimation, or even disappearance of the fast-rejoining phase. This is due to substantial rejoining of fragments taking place before the fragments are of sufficient size to be monitored.

DNA fragmentation by charged particle tracks.

High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons (60Co) or 125 keV/micrometers nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.

Stress-induced changes in brain serotonergic activity, plasma cortisol and aggressive behavior in Arctic charr (Salvelinus alpinus) is counteracted by L-DOPA.

Arctic charr (Salvelinus alpinus) were tested for aggressive behavior using intruder tests, before and after 2 days of dyadic social interaction. Following social interaction, half of the dominant and half of the subordinate fish were given L-DOPA (10 mg/kg, orally), whereas the remaining dominant and subordinate fish were given vehicle. One hour following drug treatment, the fish were tested for aggressive behavior again in a third and final intruder test, after which blood plasma and brain tissue were sampled for analysis of plasma cortisol concentrations and brain levels of monoamines and monoamine metabolites. Subordinate fish showed a reduction in the number of attacks launched against the intruder, as well as an increase in attack latency, as compared to prior to dyadic social interactions. Social subordination also resulted in an elevation of brain serotonergic activity. Fish receiving L-DOPA prior to the final intruder test showed shorter attack latency than vehicle controls. Drug treatment was a stressful experience and vehicle controls showed elevated plasma cortisol levels and longer attack latency as compared to before treatment. L-DOPA-treated fish showed lower plasma levels of cortisol and lower serotonergic activity in certain brain areas than vehicle controls. These results suggest that L-DOPA counteracts the stress-induced inhibition of aggressive behavior, and at the same time inhibits stress-induced effects on brain serotonergic activity and plasma cortisol concentrations.

Engraftment and growth of transplanted pancreatic islets.

Transplantation of pancreatic islets may provide a cure for type 1 diabetes. However, this treatment can currently be offered only to very few patients. To improve transplantation success we need to understand better the mechanisms of how the implanted islets survive, grow and/or maintain adequate function. We herein report on our studies to evaluate the factors responsible for the engraftment, i.e. revascularization, reinnervation etc., of transplanted islets and relate these factors to the metabolism and growth of the islets. Graft metabolism can be monitored by microdialysis probes that allow for the measurement of minute amounts of islet metabolites and hormonal products. Growth of the endocrine cells can be stimulated both in vitro before implantation and in vivo post-transplantation. Another problem is rejection of transplanted islets, which may be overcome by the microencapsulation of islets. The knowledge gained by the present studies will enable us to elucidate the optimal treatment of islets to ensure a maximal survival of the transplanted islets, and may be applied also to clinical islet transplantation.

DNA damage induced by radiation of different linear energy transfer: initial fragmentation.

PURPOSE: To investigate DNA fragmentation as a function of linear energy transfer (LET) after exposure to accelerated ions in the LET range 40-225 keV/microm. MATERIALS AND METHODS: Fragmentation patterns of double-stranded DNA in the range 5 kilobasepairs (kbp) to 5.7 megabasepairs (Mbp) were analysed after irradiation of low-passage GM 5758 normal human fibroblast cells with 60Co-photons, helium ions at 40 keV/microm and high-LET nitrogen ions between 80 and 225 keV/microm. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of 1-6 Mbp and 5 kbp to 1.5 Mbp fragments. RESULTS: An increased probability of formation of short and medium-sized DNA fragments was revealed following high-LET irradiation. The DNA double-strand break (dsb) induction yields were, respectively, 5.8 and 6.9-8.8 x 10(-9) dsb bp(-1) Gy(-1) for 60Co-photons and ions. The ion yields were some 80-110% higher than those calculated according to a conventional approach, disregarding the fragment distributions. For photons, the yield was 13% higher. The corresponding relative biological effectiveness (RBE) of dsb induction was in the range 1.2-1.5. CONCLUSIONS: A significant non-random contribution to the number of dsb after irradiation with high-LET was confirmed by detailed fragment analysis using pulsed-field gel electrophoresis. The LET had a strong influence on the initial DNA fragment distribution, and hence also on the induction yields measured. However, when the LET was increased to the highest values studied for nitrogen ions, the yield decreased slightly.

Rejoining of DNA fragments produced by radiations of different linear energy transfer.

PURPOSE: To analyse the rejoining of DNA double-strand breaks (dsb) produced by high-linear energy transfer (LET) ions, with the specific focus on the influence, on the rejoining estimates, of the way dsb are distributed along chromosomes. MATERIAL AND METHODS: Low passages of normal human fibroblasts (GM5758) were irradiated with 60Co photons, 40 keV/microm helium ions or nitrogen ions with LETs of 80, 125, 175 and 225 keV/microm. The double-stranded DNA fragment distributions, ranging from 5 kbp to 5.7 Mbp, were assayed by pulsed-field gel electrophoresis after repair incubation for 0-22 h. RESULTS: The rejoining was biphasic and the half-times of the two phases were 15 min and 2-3h, respectively, and were independent of LET. Although the majority of breaks were rejoined by the fast phase, both the fraction of dsb rejoined by the slow phase and the fraction of unrejoined dsb at 20-22h increased with increasing LET. CONCLUSIONS: DNA fragment analysis detected LET-dependent differences in the amount of rejoining while the half-times were independent of LET. The majority of dsb were rapidly rejoined even after high-LET irradiation. If fragment-size distribution is not taken into account, both the fraction of breaks rejoined by slow kinetics, and the fraction of unrejoined breaks, can be overestimated when the LET is increased.

Skin darkening, a potential social signal in subordinate arctic charr (Salvelinus alpinus): the regulatory role of brain monoamines and pro-opiomelanocortin-derived peptides.

Arctic charr were allowed to interact in groups of three for 5 days. Skin darkness was quantified by measuring the mean brightness of individual fish before and after social interaction. Brain levels of monoamines and monoamine metabolites and plasma concentrations of cortisol, adrenocorticotropic hormone (ACTH), N-acetyl-(beta)-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) were analysed. The results show that social subordination resulted in a significant skin darkening. Furthermore, plasma concentrations of alpha-MSH, ACTH and cortisol were elevated in subordinates, and these fish also displayed elevated levels of 5-hydroxyindoleacetic acid (5-HIAA) in the telencephalon. The ratio of [5-HIAA] to serotonin [5-HT] was increased in several brain areas. In addition, the ratio of 3-methoxy-4-hydroxyphenylglycol (MHPG) to norepinephrine (NE) concentrations was significantly increased in the optic tectum of subordinate fish. Skin darkness following social interaction showed a significant positive correlation with plasma levels of alpha-MSH. Plasma levels of ACTH and alpha-MSH were both positively correlated with that of cortisol. Brain [5-HIAA]/[5-HT] ratios were positively correlated with circulating plasma levels of ACTH, and a similar positive correlation was seen between [MHPG]/[NE] ratios in the optic tectum and plasma levels of ACTH, alpha-MSH and N-acetyl-beta-endorphin. In contrast, hypothalamic [MHPG]/[NE] ratios displayed a negative correlation with plasma alpha-MSH concentrations. The present study demonstrates that social stress induces skin darkening in Arctic charr and that this effect could be mediated by a stress-induced increase in the levels of alpha-MSH in the circulation. Furthermore, the results suggest that 5-HT and NE in the central nervous system could be factors regulating the pituitary release of ACTH and alpha-MSH.

Differential effects on T cell and NK cell development by tissue-specific expression of H-2D(d) transgene.

The effect of tissue-specific expression of the MHC class I molecule H-2D(d) on T cell and NK cell specificity was studied in transgenic mice expressing the H-2D(d) gene under the control of the mouse metallothionein-I promoter. MTD mice expressed high amounts of H-2D(d) in the liver, intestine and testis, but only minute amounts in the thymus, spleen and kidney. Zinc administration resulted in a 1.5- and 8.5-fold increase in H-2D(d) expression in the liver and the intestine, respectively, but did not affect expression in the other organs tested. T cell tolerance developed towards H-2D(d) in MTD mice, even in the absence of zinc. In contrast, NK cell-mediated natural resistance against lymphoma grafts was not seen in MTD mice, despite zinc administration. NK cells in MTD mice also failed to develop self tolerance to H-2D(d). The lack of functional effects did not result from inability of NK cells in MTD mice to interact with H-2D(d), as down-regulation of Ly49A receptor expression was observed on liver NK cells in MTD mice. Our data reveal a difference between T cells and NK cells in their requirements for MHC class I molecules in specificity development.

Induction and rejoining of large DNA fragments after ion irradiation.

Radiation-induced DNA double-strand breaks (DSBs) were analyzed by separating large DNA fragments by pulsed-field gel electrophoresis. Human U-343MG glioma and K562 erythroleukemia cells were irradiated with 60Co gamma rays or nitrogen ions with high linear energy transfer (125 keV/microm). By comparing the fraction of DNA released into the gel below different size thresholds, corresponding to megabase-pair-sized DNA fragments, the relative effectiveness of the nitrogen ions was found to be dependent on both dose and the threshold size used in the evaluation. This dose dependence was most evident for the smallest threshold (6 Mbp) and was due to a linear dose response for release of the fragments for the ions compared to the curvilinear response for the gamma rays. The two curves intersected, and the relative yield of fragments (nitrogen ions/gamma rays) decreased from more than 3 below 1.5 Gy to 0.8 at 30 Gy. For the larger sizes (6-10.5 Mbp), the relative yield was constant at around 0.7. Thus the ion-induced fragments were shifted to smaller sizes compared to the 60Co gamma rays, and the data for nitrogen ions could not be fitted to random fragment distributions at doses < or =20 Gy. From these results, we conclude that a substantial fraction of the DSBs induced by heavy ions were nonrandomly distributed, correlated with DSBs within a region of < or =2 Mbp. After a dose of 20 Gy, the rejoining curves for ion-induced DSBs were different for each fragment size, resulting in different levels of unrejoined breaks after 6 h.

Alpha1/alpha2 domains of H-2D(d), but not H-2L(d), induce "missing self" reactivity in vivo--no effect of H-2L(d) on protection against NK cells expressing the inhibitory receptor Ly49G2.

Introduction of the MHC class I transgene H-2Dd on C57BL/6 (B6) background conveys NK cell-mediated "missing self" reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2Dd/Ld MHC class I transgenes, and show that the alpha1/alpha2 domains of H-2Dd were necessary and sufficient to induce "missing self" recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the alpha1/alpha2 domains of H-2Ld induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the alpha1/alpha2 domains of H-2Ld on NK cell specificity was surprising, considering that both H-2Ld and H-2Dd have been reported to interact with Ly49G2. Therefore, the role of H-2Ld for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2Dd, but not H-2Ld, abolished killing by sorted Ly49G2+ NK cells, indicating that H-2Ld does not inhibit NK cells via the Ly49G2 receptor.