Consequences of acute stress and cortisol manipulation on the physiology, behavior, and reproductive outcome of female pacific salmon on spawning grounds

Contents lists available at Hormones and Behavior Consequences of acute stress and cortisol manipulation on the physiology, behavior,and reproductive outcome of female Pacific salmon on spawning grounds Sarah H. McConnachie ,, Katrina V. Cook , David A. Patterson , Kathleen M. Gilmour , Scott G. Hinch Anthony P. Farrell , Steven J. Cooke a Fish Ecology and Conservation Physiology Laboratory, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6b Fraser Environmental Watch Program, Fisheries and Oceans Canada, Pacific Region, Science Branch, Cooperative Resource Management Institute,School of Resource and Environmental Management, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6c Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5d Department of Forest Sciences and Institute of Resources, Environment and Sustainability, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4e Department of Zoology and Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4f Institute of Environmental Science, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 Life-history theory predicts that stress responses should be muted to maximize reproductive fitness. Yet, the Received 20 December 2011 relationship between stress and reproduction for semelparous salmon is unusual because successfully Revised 30 April 2012 spawning individuals have elevated plasma cortisol levels. To tease apart the effects of high baseline cortisol Accepted 2 May 2012 levels and stress-induced elevation of cortisol titers, we determined how varying degrees of cortisol elevation Available online 8 May 2012 (i.e., acute and chronic) affected behavior, reproductive physiology, and reproductive success of adult femalepink salmon (Oncorhynchus gorbuscha) relative to different states of ovulation (i.e., ripe and unripe). Exhaus- tive exercise and air exposure were applied as acute stressors to manipulate plasma cortisol in salmon either confined to a behavioral arena or free-swimming in a spawning channel. Cortisol (eliciting a cortisol eleva- tion to levels similar to those in post-spawn female salmon) and metyrapone (a corticosteroid synthesis in- Oncorhynchus gorbuscha hibitor) implants were also used to chemically manipulate plasma cortisol. Cortisol implants elevated plasma Hormone injections cortisol, and impaired reproductive success; cortisol-treated fish released fewer eggs and died sooner than fish in other treatment groups. In contrast, acute stressors elevated plasma cortisol and the metyrapone im- plant suppressed plasma cortisol, but neither treatment significantly altered reproductive success, behavior, or physiology. Our results suggest that acute stressors do not influence behavior or reproductive outcome when experienced upon arrival at spawning grounds. Thus, certain critical aspects of salmonid reproductioncan become refractory to various stressful conditions on spawning grounds. However, there is a limit to theability of these fish to tolerate elevated cortisol levels as revealed by experimental elevation of cortisol.
2012 Elsevier Inc. All rights reserved.
an emergency response and animals attempt to regain Considerable evidence supports the notion that stress can impair allostasis (). Yet, much of the existing the reproductive outcome of a wide range of vertebrates, including work on chronic stress/glucocorticoid elevation is focused on the birds ), reptiles long-term consequences for animals during non-reproductive pe- riods rather than immediately before or during reproduction. For ex- ), and fish ). The ample, many toxicological studies demonstrate direct long-term acute stress response and associated elevation of glucocorticoids is reproductive impairments (e.g., suppression of reproductive hor- believed to be adaptive, while chronic elevation of glucocorticoids mones) associated with emergency resource reallocation to mainte- can have various negative tertiary effects, including impaired im- nance and survival (e.g., reviewed in see mune function and fitness whenever resources are directed towards also ).
Furthermore, most of these studies consider iteroparous species (i.e.
repeat breeders), which have the life-history option of delaying a ⁎ Corresponding author.
reproductive event when challenged.
E-mail addresses: (S.H. McConnachie), In contrast, semelparous species usually cannot delay the repro- (K.V. Cook), (D.A. Patterson), ductive event because they invest in reproduction only once in a life- (K.M. Gilmour), (S.G. Hinch),(A.P. Farrell), (S.J. Cooke).
time. For semelparous fishes such as Pacific salmonids (Oncorhynchus 0018-506X/$ – see front matter 2012 Elsevier Inc. All rights reserved.
doi: S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 spp.), some argue that the spawning date is genetically fixed, which successfully blocks cortisol synthesis in fish in the short-term implies that it cannot be altered by external stressors Curiously, virtually nothing is known about whether exposing but has rarely been used with a cocoa butter carrier (but see semelparous Pacific salmonids to stress on spawning grounds influ- ). Rainbow trout (O. mykiss), a congeneric of pink ences their behavior and reproductive success. Yet, these fish routine- salmon, weighing approximately 150 g were anesthetized with ben- ly encounter many stressors that trigger a cortisol response as they zocaine (0.05 mg ml−1 water; p-aminobenzoic acid ethyl ester; approach their spawning date, suggesting that the acute stress re- Sigma E1501, Sigma-Aldrich) and given an IP injection of metyrapone sponse remains active during the reproductive period. For example, mixed in heated liquid cocoa butter (200 mg kg−1 fish in l ml cocoa plasma cortisol rises when fish encounter hydraulic challenges and butter kg−1 fish); upon injection into the fish, the cocoa butter rapid- elevated water temperature during the spawning migration ( ly cools to a thick paste, providing a slow-release metyrapone im- ). Furthermore, a progressive increase plant. After 1 and 5 days, fish were subjected to 1 min of air in baseline plasma cortisol levels of unknown etiology occurs as salm- exposure as an acute stressor, and a blood sample was withdrawn on swim to the spawning grounds by caudal puncture 30 min later for assessment of plasma cortisol levels. The expectation was that this 30-min delay would be sufficient cortisol concentrations rise from 25 ng ml−1 in pink salmon (O.
for the maximum or near maximum rise in plasma cortisol level to be gorbuscha) at river entry (), to 350 ng ml−1 on arrival at the spawning ground (female sockeye salmon [O. nerka];), and 1287 ng ml−1 when the fish become mor- Weaver Creek spawning channel ibund (female sockeye salmon; ). Thus, an acutestressor can elevate plasma cortisol against a background of progres- All field experiments were conducted at the Weaver Creek sively increasing plasma cortisol levels during the spawning spawning channel located in British Columbia, Canada (see for detailed information). Each experiment involved A stressed state should generally be incompatible with reproduc- groups of naive fish (i.e., fish were not reused among experiments).
tion and, based on life-history theory, one could postulate that the The artificial channel, 2.93 km long and 6.1 m wide, is composed of cortisol stress response of semelparous salmon should be muted, or a cobble (1.2–7.6 cm) substrate and has a consistent water depth of physiologically irrelevant, during this period ( 25–30 cm. Fish densities and flow conditions were monitored ) to mitigate any potential negative effects of cortisol throughout the spawning period and manually operated gates were elevation above the (high) baseline levels on spawning grounds.
used to regulate fish movements into the spawning channel Thus, we postulate that reproductive drive in a semelparous salmon (). Experiments were timed to coincide with peak species will outweigh any cortisol-mediated mating inhibition.
pink salmon spawning activity in early October 2009.
Acute, stress-related increases in plasma cortisol suppress the normalincreases in plasma sex hormone concentrations for Pacific salmon Reproductive physiology on arrival during early phases of upriver migration (However,increases in plasma cortisol during migration are regarded as adap- On arrival at the spawning channel in early October, female pink tive and necessary for salmon to be able to return to their natal salmon were individually removed from the raceway via dip net streams and spawn (Complicating matters is and immediately placed in a trough supplied with flow-through the fact that spawning Pacific salmon also undergo senescence, water from the raceway. Fish were categorized as either "unripe" which alters many physiological processes, including hormone regu- (N = 52, unovulated, where eggs are still confined to intact ovaries) lation To address or "ripe" (N = 60, ovulated, where eggs have been released into the these issues, we experimentally determined how short-term changes body cavity and gentle abdominal pressure near the vent easily ex- in and experimental manipulation of plasma cortisol influenced the pels eggs) and a blood sample was collected via caudal puncture reproductive physiology, behavior, and spawning outcome of wild fe- (2 ml blood sample; collected using 3 ml vacutainer and 1.5 in., male pink salmon (O. gorbuscha). We administered cortisol implants 18 ga needle, lithium heparin; Becton Dickson, NJ) within 30 s and predicted that plasma cortisol elevation, lasting between 2 and (Within 3 min the fish were released back into 5 days, would negatively affect reproductive behavior (e.g., less time the spawning channel. Blood samples were stored in an ice-water spent guarding eggs or fighting for a mate), physiology (i.e., suppres- slurry and centrifuged (5 min at 10,000 g) within 45 min, after sion of reproductive hormones), and outcome (i.e., number of eggs which the plasma was frozen in liquid nitrogen immediately. Samples released). We also predicted that the response to acute stressors were subsequently stored at −80 °C until further analysis.
(i.e., exhaustive exercise or air exposure) would be muted in semel- In addition, subsets of ripe (N= 6) and unripe (N= 12) salmon parous salmon and would not alter these same responses. Conversely, were given an intraperitoneal (IP) injection of either cortisol (hydrocor- an intraperitoneal (IP) implant of metyrapone, which blocks the last tisone 21-hemisuccinate; Sigma H4881, Sigma-Aldrich; 110 mg kg−1 step of glucocorticoid synthesis, was expected to lower plasma corti- fish in 50 ml melted cocoa butter kg−1 fish; ) to el- sol levels () and retard reproduction and senes- evate cortisol levels for a short period (i.e., 2 to 5 days), or metyrapone cence. To our knowledge, hormone manipulations of this type had (200 mg kg−1 fish; 1 ml cocoa butter kg−1 fish) to block glucocorticoid not before been performed on senescing Pacific salmon.
synthesis (), before being placed in individual,opaque, experimental chambers ( 50 l) situated on the bank of the Materials and methods channel and equipped with flow-through water. Fish were leftundisturbed for approximately 24 h, after which they were individually Metyrapone validation removed and blood was sampled immediately via caudal puncture.
All fish were handled in accordance with the guidelines of the Ca- Longevity and reproductive status study nadian Council on Animal Care (Carleton University, B09-12; Univer-sity of Ottawa, BL-228). A pilot laboratory experiment was carried out On October 6th and 7th 2009, 120 unripe pink salmon that had to determine the effectiveness of metyrapone (2-methyl-1, 2-di-3- voluntarily entered the raceway were marked with unique individual pyridyl-1-propanone; Sigma 85625, Sigma-Aldrich) at blocking corti- Peterson disk tags placed in the dorsal musculature. The tags could be sol synthesis when delivered in a cocoa butter implant. Metyrapone read on free-swimming fish with binoculars, which allowed the fish S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 to be observed without any disturbances. Fish were randomly fish was on the receiving end of an aggressive act (both were assigned to one of six treatment groups (N = 20 per treatment summed and divided by total observation minutes, and aggression re- group): a) control fish (only tagged); b) sham injection-controls ceived was subtracted from aggression given to yield an overall ag- (tagged and given an IP injection of 50 ml kg−1 melted cocoa butter); gression score). The daily duration of behavioral observations on c) cortisol-treated (as described above); d) metyrapone-treated (as each fish (i.e., 10 min) was consistent with other studies described above); e) chased (acutely stressed by 3-min of being and is believed to be representative of longer time periods "chased" by hand around a circular tank supplied with flow-through given the reasonable predictability and stability of behavioral reper- channel water); and f) air-exposed (as in (e), followed by 1 min of toires for this species. After 4 days, the fish were collectively culled air exposure to increase the severity of the acute stressor). After- in a process lasting b 10 min; fish were killed by cerebral percussion.
wards, fish were immediately released into the spawning channel After immediate blood sampling, the percentage of eggs released and closely monitored during daylight hours so that moribund or was estimated (as described above).
dead fish could be collected daily.
Longevity in the spawning channel following release (i.e. time until death after arrival) was calculated using the methods outlined inFork length, total mass, gonad mass, epidermal Plasma glucose and cortisol concentrations were measured as in- coverage by fungus, and general condition also were documented. Re- dicators of stress Briefly, plasma glucose productive status was reported as the percentage (%) of eggs released values were determined using a YSI 2300 STAT Plus glucose analyzer by each individual. The relationship between percentage of eggs (YSI Inc., Yellow Springs, Ohio). Plasma cortisol levels were measured remaining relative to percentage of eggs initially expected was deter- using a commercial ELISA kit (Neogen Corporation # 402710, Lexing- mined following the methods of . Briefly, the antic- ton KY). For cortisol, the assay has 47% cross-reactivity with the drug ipated initial gonad mass was determined from a known relationship prednisolone, which would not be present in the samples.
between body mass and gonad mass established for a separate group The assay also has 15% cross-reactivity with cortisone and 11- of mature, unripe pink salmon sampled from the spawning channel deoxycortisol. The analytical sensitivity (B/B (N= 21; gonad mass= 10.1·body mass −297.9, R2=0.80, P=0.005).
0, 80%) for the cortisol assay was at 0.04 ng ml−1. Testosterone and 17β-estradiol are both Eggs were weighed and counted in whole ovaries and a linear body major reproductive hormones and plasma concentrations of these hor- mass to fork length relationship, together with a linear fork length to mones also were measured by ELISA kits (Neogen Corporation, gonad mass relationship, was used to interpolate the expected egg , catalog numbers: 402110, 402510). Testosterone and mass before ovulation for the experimental fish. Many fish had 17β-estradiol were extracted from plasma samples using ethyl ether spawned all of their eggs (100% success), but any eggs remaining according to the kit manufacturer's protocols. The assay manufacturer were weighed first as five groups of 10 eggs, with any eggs remaining states that the estradiol assay does not cross-react with any other estro- thereafter being weighed collectively. Individual egg mass is known to gens. Analytical sensitivity (B/B be uniform within an individual (D. Patterson, personal communica- 0, 80%) was at 0.03 ng ml− 1. According to the manufacturer, the testosterone assay is 100% cross-reactive tion), and so this method provided an accurate estimate of the number with dihydrotestosterone and the analytical sensitivity (B/B of eggs retained by each fish without having to count every egg.
was at 0.006 ng ml−1. Cortisol, glucose, testosterone, and 17β-estradiol were assayed in duplicate at appropriate dilutions. Inter- and Spawning behavior in enclosures intra-assay variability was b10% for all assays. More detailed descrip-tions of the analytical techniques can be found in Behaviors were studied in unripe and ripe salmon held in enclo- sures that had been constructed within the spawning channel. Ablood sample (as described above) was withdrawn from 30 salmon(6 treatment groups as above; N = 5 for each treatment group) in Statistical analysis the raceway before placing them in a holding tank for transfer to asection of the spawning channel that housed a net-pen (2 m wide Results from the metyrapone pilot study were analyzed using a by 15 m long; constructed out of Vexar rigid mesh fencing; Master- two-way analysis of variance (ANOVA) to determine whether cortisol net, Mississauga, Ontario). Fish were treated according to their exper- values varied by treatment and time. Results from the cortisol and imental group before being placed into the enclosure. Twenty "ripe" metyrapone validation study before and after 24 h were compared male pink salmon (i.e. males that released sperm when squeezed using two-way repeated measures ANOVA models with time and gently near the vent) had been placed into the net-pen 12 h earlier.
treatment as effects. For the channel experiment, longevity among Fewer males were placed in the pen than females to facilitate compe- treatment groups was compared using a log-rank survival analysis tition among females. Two trials were completed for unripe salmon in to 50% mortality. The percentage of eggs released by each fish was early October 2009, and two trials were completed for ripe fish in late averaged within groups and compared using a one-way ANOVA. For October 2009.
the enclosure experiments, all hormone and blood physiology values Behavioral observations were carried out for 10 min daily on four and behavioral metrics were compared before and after 4 days using consecutive days. The order of observing each fish was randomized two-way repeated measures ANOVA models with time and treatment daily. Reproductive behaviors of pink salmon are well known, and being the independent variables. Time until territory establishment are similar to behaviors displayed by other semelparous Pacific salm- was determined using log-rank survival analyses. The percentage of on Females prepare their eggs released by each fish was averaged for each treatment group and nesting area, fend off intruders from their territory through aggres- compared using a one-way ANOVA. Tukey's post-hoc tests were sive action, and spend time with males to ensure fertilization occurs.
employed following significant one-way ANOVAs to determine differ- We recorded on what day fish established a territory, how much time ences among groups (where p b 0.05). The assumptions of equality of the fish spent holding that territory (represented as a percent, aver- variances and normal distribution were tested for all analyses and aged over days on territory), what percentage of their time females relevant transformations applied where assumptions could not be met.
spent with males (averaged across days on an established territory), Percentage data were arcsine transformed prior to analysis. Where trans- the number of nest construction digging behaviors that occurred (av- formation of the data was not possible or effective, non-parametric anal- eraged across days spent on a territory), how many times a fish made yses were performed. All analyses were conducted using JMP, version an aggressive display towards a conspecific, and how many times that 8.0.2 (SAS Institute Inc., Cary, NC). The level of significance (α) for all

S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 tests was assessed at 0.05. All data are presented as mean ±standard error unless otherwise noted.
Initial blood hormone and glucose values of ripe and unripe pink salmon(Oncorhynchus gorbuscha) removed from the Weaver Creek raceway in October,2009, presented as mean (± SE). N = 52 for unripe fish and N = 60 for ripe fish. All data were analyzed using the Wilcoxon Rank-Sum Test, except for cortisol (*), whichwas analyzed using log-transformed data in a one-way ANOVA.
Effectiveness of metyrapone Metyrapone-treated rainbow trout subjected to an acute stressor exhibited significantly lower plasma cortisol concentrations than Glucose (mmol l−1) sham-treated fish 1 day following treatment (two-way ANOVA Time Cortisol (ng ml−1)* Estradiol (ng ml−1) effect: F = 7.8, df = 1, p = 0.02; but not after 5 days (Treat- Testosterone (ng ml− 1) ment effect: F = 3.1, df = 1, p = 0.1; Interaction: F = 4.7, df = 3,p = 0.03; ). Therefore, we assumed that pink salmon would ex-perience a short-term depression of plasma cortisol during acute measures ANOVA: Treatment effect: F= 1.0, df =1, p b 0.001; Time: stress (i.e., for at least 24 h but not as long as 5 days) and used F = 34, df =1, pb 0.001; Interaction: F=5.4, df=3, pb 0.001; cocoa butter as a vehicle for metyrapone delivery.
Plasma glucose values increased 24 h after either treatment (Treatmenteffect: F =5.1, df =1, p= 0.3; Time: F =6.8, df =1, p= 0.02; Interaction: Raceway blood physiology and hormone validations F= 2.2, df =3, p=0.03; Estradiol was unaffected by either treat-ment (Treatment effect: F=0.69, df= 1, p=0.4; Time: F= 0.90, df =1, Reproductive hormone titers were indicative of whether pink salmon in the spawning channel were ripe or unripe (). Plasma estradiol and testosterone were both signifi-cantly lower in ripe fish (estradiol: F = 70, df = 1, p b 0.001; testoster-one: F = 25, df = 1, p b 0.001; However, plasma cortisolconcentrations were similar (one-way ANOVA, F = 0.31, df = 1,p = 0.6; and plasma glucose concentrations were higher inripe fish (one-way ANOVA, F = 13, df = 1, p b 0.001; ) for arriv-ing pink salmon.
For unripe fish held in isolation chambers, cortisol implants signifi- cantly elevated plasma cortisol by 10-fold, but metyrapone implantshad no effect on circulating cortisol levels after 24 h (two-wayrepeated-measures ANOVA: Treatment effect: F=55, df= 1, p b 0.001;Time: F=70, df= 1, p b 0.001; Interaction: F=15, df=3, pb 0.001;Plasma glucose was unchanged 24 h after either treatment(Treatment effect: F=0.69, df =1, p =0.4; Time: F= 0.90, df =1,p= 0.4; Interaction: F= 0.39, df =3, p =0.9; ). Plasma concentra-tions of both estradiol (Treatment effect: F =0.8, df= 1, p =0.8; Time:F= 8.5, df =1, p =0.02; Interaction: F=1.5, df= 3, p= 0.3; C)and testosterone (Treatment effect: F=0.13, df= 1, p =0.7; Time:F= 5.7, df =1, p=0.04; Interaction: F=1.7, df= 3, p =0.2; D) de-creased 24 h after either treatment.
For ripe fish held in isolation chambers, the cortisol implant again increased plasma cortisol values, but the response was attenuated com-pared with that of unripe fish E). Plasma cortisol concentrationwas not affected by the metyrapone implant (two-way repeated- Fig. 2. A–H. Summary of pink salmon (Oncorhynchus gorbuscha) plasma hormone and Fig. 1. Mean (± SE) cortisol values for control and metyrapone-treated rainbow trout glucose values for unripe (A–D) and ripe (E–H) fish both before and 24 h after treat- (Oncorhynchus mykiss) subjected to an air-exposure stressor either 1 or 5 days after ment with cortisol or metyrapone. Values are stated as mean (± SE). Dissimilar letters treatment with metyrapone. Data were log-transformed and analyzed using a two- denote significant differences among treatment groups and time periods (Tukey– way ANOVA. Dissimilar letters denote a significant difference between treatment Kramer HSD test, p b 0.05). N = 6 for each treatment for unripe fish; N = 12 for ripe groups and/or time periods (Tukey–Kramer HSD test, p b 0.05). Sample sizes are as fol- fish. All Ranked Sum data were analyzed using a two-way repeated-measures lows: 1 day: control = 2, metyrapone = 5. 5 days: control = 4, metyrapone = 4.
ANOVA, with time and treatment as independent variables.

S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 p= 0.4; Interaction: F=1.2, df= 3, p =0.3; Plasma testosteronewas decreased 24 h after both treatments (Treatment effect: F=0.83,df= 1, p= 0.4; Time: F =21, df =1, pb 0.001; Interaction: F=0.27,df= 3, p= 0.6; Longevity and reproductive study Pink salmon treated with cortisol exhibited reduced longevity rel- ative to all other treatment groups (log-rank survival time to 50%mortality; λ2 = 13.1, df = 5, p = 0.02; ). Cortisol-treated fishalso released fewer eggs during their time in the channel comparedwith all other treatment groups except the sham group [47% forcortisol-injected; 69% for sham-treated; >85% for all other groups(one-way ANOVA, F = 13, df = 5, p b 0.001; )].
Enclosure experiment: reproductive status Fig. 4. A comparison across treatment groups (see text for details of treatment groups)of the percentage (%) of total possible eggs deposited by pink salmon (Oncorhynchus Treatment with cortisol, metyrapone or acute stress did not influ- gorbuscha) in the Weaver Creek spawning channel during early October, 2009. All ence the extent to which fish ripened during the experiment data were transformed into ArcSine (square root) values before analysis. Samplesizes were as follows; chase and control = 20, cortisol = 18, chase + 1 min air exposure . For those fish that did ripen during the enclosure experiment, and metyrapone = 17 and sham = 14. Dissimilar letters denote significant differences differences in egg release (%) were observed (Wilcoxon Rank Sum; among treatment groups (Tukey–Kramer HSD test, p b 0.05).
λ2=11.2, df=5, p=0.04; . Control and chased fish releasedmore than 80% of their eggs, chase + 1 min air exposure and cortisol-treated fish released approximately 50% of their eggs, and metyrapone Time: F = 72, df = 1, p b 0.001; Interaction: F=0.6, df=5, p=0.7; and sham-treated fish released the fewest eggs (b10%). For ripe fish, C), whereas plasma testosterone concentrations remained there were no statistically significant differences in egg release unchanged over the 4 day experimentation period (Treatment effect: among treatment groups (data not shown). However, cortisol-treated F = 1.1, df = 1, p = 0.4; Time: F = 3.3, df = 1, p = 0.08; Interaction: fish released 50% of their eggs, whereas all other treatment groups F = 0.3, df = 5, p = 0.9; D).
released >70% of their eggs.
For ripe fish, plasma cortisol levels varied across treatment groups after 4 days Control fish exhibited the highest levels (1756± Enclosure experiment: hormone profiles 274 ng ml−1), and cortisol-treated fish displayed similar concentrations (averaging 1592 ±207 ng ml−1). Values were similar among sham- Among unripe fish, cortisol-treated fish exhibited elevated cortisol treated (1118 ±211 ng ml−1), chased fish (846±289 ng ml−1), and concentrations 4 days following treatment (two-way repeated- chased+ 1 (757 ±186 ng ml−1) fish, whereas the lowest value (577± measures ANOVA: Treatment effect: F = 4.6, df = 1, p = 0.002; Time: 193 ng ml−1) was observed in metyrapone-treated fish (two-way F = 0.4, df = 1, p = 0.9; Interaction: F = 2.5, df = 5, p = 0.04; .
repeated-measures ANOVA: Treatment effect: F=2.7, df =1, p =0.03; Plasma glucose concentration increased in all fish during the 4 day ex- Time: F= 55, df =1, pb 0.001; Interaction: F=3.2, df=5, p=0.01; periment (Treatment effect: F = 0.7, df = 1, p = 0.6; Time: F = 15.6, E). Plasma glucose concentrations increased during the 4 day peri- df= 1, p b 0.001; Interaction: F=0.6, df=5, p=0.7; B). Plasma od, across treatments (Treatment effect: F=1.7, df= 1, p=0.1; Time: estradiol levels decreased (Treatment effect: F = 0.5, df= 1, p = 0.8; F= 5.4, df =1, p= 0.02; Interaction: F =0.93, df =5, p= 0.5; Plasma estradiol levels were low and did not change (Treatment effect:F= 1.2, df=1, p=0.3; Time: F=2.5, df =1, p =0.1; Interaction:F= 0.7, df =5, p =0.6; , whereas plasma testosterone concentra-tions decreased after 4 days (Treatment effect: F= 0.4, df= 1, p= 0.8;Time: F= 91, df= 1, p b 0.001; Interaction: F=0.2, df=5, p=0.9;H).
Enclosure experiment: behavior observations Among unripe fish, treatment did not influence the rate of territory establishment (log-rank survival analysis; λ2=2.4, df=5, p=0.8).
Based on behavioral observations for fish on territories, cortisol-treated fish spent 10% less time holding their territory comparedwith controls (one-way ANOVA: F = 12, df = 5, p = 0.03; Addi-tionally, cortisol-treated fish were less aggressive and experiencedmore aggressive acts by conspecifics (F=13, df=5, p=0.04;Among ripe fish, no differences were noted for territory estab-lishment (log-rank survival analysis; λ2=4.0, df=5, p=0.5). In addi-tion, no behavioral differences were observed among the treatmentsgroups ().
Fig. 3. Log-rank survival analysis to 50% mortality in each treatment group (see text for By experimentally elevating plasma cortisol in unripe fish for be- details of treatment groups), comparing longevity among pink salmon (Oncorhynchus tween 2 and 5 days with a cortisol in cocoa butter implant, we negative- gorbuscha) in the Weaver Creek spawning channel. Sample sizes were as follows:chase and control = 20, cortisol = 18, chase + 1 and metyrapone = 17 and sham = 14.
ly impacted the longevity, reproductive behavior, and reproductive

S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 Fig. 5. (A and B). Figure A presents the percentage of pink salmon (Oncorhynchus gorbuscha) that became ripe during the behavior trials and thus were able to spawn during theenclosure experiment. Figure B presents the percentage of total eggs available (%) that were deposited during the 4 day trials by ripened fish across treatment groups. Sample sizeswere as follows: chase = 1/9, chase + 1 = 2/8, control = 3/9, cortisol = 3/10, metyrapone = 2/9, sham = 4/8. All data were transformed into ArcSine (square root) values beforebeing analyzed. Dissimilar letters denote significant differences among treatment groups (Tukey–Kramer HSD test, p b 0.05).
outcome of pink salmon on their spawning grounds. Conversely, acute Nonetheless, collectively these data are consistent with the notion stressors that also presumably elevated plasma cortisol, namely exercise that semelparous salmon may be resilient to the effects of stress hor- and air exposure, did not affect reproductive outcomes in either ripe or mones during the final phases of reproduction unripe fish. These results demonstrate that a sustained elevation of plas- ). However, in the case of Pacific salmon, it is unclear ma cortisol carries significant reproductive costs for semelparous salmon when such a transition takes place during the migration. In main- on their spawning grounds (despite their high baseline cortisol levels), stream riverine habitats, fish mount a cortisol response to a stressor but that temporary elevations may not. In an ecologically relevant and cortisol does, indeed, result in suppression of reproductive context, events that could elicit a prolonged stress response that might hormone titers ). Yet, our data indi- last 2–5 days include periods of high water temperature ( cate that, upon arrival at spawning grounds, reproductive hormones are ), seasonally high (or low) river discharge ), not altered by either certain acute stressors that are expected to elevate river obstructions or regions that are hydraulically complex ( plasma cortisol levels (see below) or experimental cortisol manipulation.
), or disease ). In contrast, very short- Because we did not observe any differences between ripe and unripe fish term stressors, which might include fisheries interactions, failed preda- with respect to the influence of cortisol elevation on hormone titers, the tion events, and antagonistic interactions with conspecifics just prior to onset of resistance to elevated cortisol appears to occur prior to ovulation, or during spawning may result in fewer effects on reproduction.
a point that warrants investigation in a further study. The transition maybe associated with the decline from stable levels of reproductive hor- Cortisol manipulation and reproductive hormones mones as the fish move into an ovulated state. During ovulation, thereis a critical need to increase 17α-hydroxy-20 β-dihydroprogesterone In a variety of fish species, elevation of glucocorticoids results in de- (17α, 20β-P) to complete reproduction because this creased reproductive hormone concentrations (see review by hormone induces sexual maturation necessary for the ovulation process, ), which in iteroparous fish can lead to a postponed whereas estradiol and testosterone mediate maturation and ovulation reproductive event. Additionally, a stressful reproductive environment (e.g., fish exposed to bleached kraft pulp mill effluent) negativelyimpacts reproductive fitness in various ways Channel longevity and reproductive success ). In semelparous Pacific salmonexposed to a natural hydraulic challenge during their reproductive Cortisol-treated fish exhibited decreased longevity and high egg migration up the Fraser River system in BC (at the Hell's Gate fishway, retention during the channel experiment, despite our finding that in the Thompson Canyon, BC, ), cortisol treatment did not change reproductive hormone titers.
reproductive hormone titers (i.e., 11-ketotestosterone, estradiol and Therefore, chronic cortisol elevation on spawning grounds negatively testosterone) fall dramatically while cortisol levels increase. Further influences reproductive function and success. Even though egg re- upstream, where the river is less challenging and perhaps less than lease by metyrapone- and sham-treated fish during the unripe enclo- 1 day later in the migration, baseline values of cortisol are restored sure experiment was reduced when compared to other treatment ( 100 ng ml−1) and reproductive hormones return to their elevated groups, overall these fish still released the majority of their eggs and levels Yet prior to the present study, the potential longevity was comparable to control groups. As such, even if the interactions between cortisol and reproductive hormone oscillations cocoa butter implant did prevent some egg release in the sham, corti- had not been investigated in terms of impacts on behavior at spawning sol, and metyrapone treatments (see discussion below), the existence grounds and reproduction for a semelparous species. The raceway of differences among these treatments lends support to the notion blood profiles and hormone validation data collected in the present that the driver of the differences was of a physiological nature rather study indicated that, even though cortisol titers in cortisol-treated than an artifact of the use of cocoa butter.
fish were increased to levels observed in senescing salmon ( This suite of findings is particularly important because fisheries managers are concerned with the largely unexplained phenomenon cortisol treatment did not alter reproductive hormone titers in either of "pre-spawn mortality"—fish that die on spawning grounds either unripe or ripe fish.
without spawning or with significant egg retention It is important to recognize that the experimental elevation of cor- The eggs of such fishes are often still viable ( tisol titers with IP implants is not itself a stress response, but instead so it appears that other factors are inhibiting reproductive be- results in elevated cortisol that is consistent with a stress response.
havior and/or are advancing senescence. In a study of sockeye salmon

S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 Fig. 6. (A–H). Blood hormone and glucose values of unripe (A–D) and ripe (E–H) pink salmon (Oncorhynchus gorbuscha) before experimentation and 4 days after treatment (seetext for treatment details), stated as mean values (± SE). All Ranked Sum data were analyzed using two-way, repeated-measures ANOVAs with time and treatment as the indepen-dent variables. Dissimilar letters denote significant differences among treatment groups and time periods (Tukey–Kramer HSD test, p b 0.05). Sample sizes were as follows for un-ripe fish: before; cortisol = 10, control, chase and metyrapone = 9, chase + 1 and sham = 8. After; cortisol = 10, chase and control = 9, chase + 1, sham and metyrapone = 8. Samplesizes were as follows for ripe fish: N = 10 for all groups except for the "after" chase group where N = 9.
Table 2Pink salmon (Oncorhynchus gorbuscha) behavior profiles for unripe fish during 4 day trials; values are stated as mean (±SE). All data were analyzed using Wilcoxon Rank-Sumtests, and Tukey's HSD test was used to determine where differences lay when a significant effect was obtained (noted by letter scores). All data that are expressed as percentageswere transformed into ArcSine (square root) values before being analyzed. Data for all variables except the aggression score were averaged over days that fish were on establishedterritories. Aggression scores were added for all days spent on territories and divided by number of observational min. Each fish had a similar score for aggressive attacks against,and this score, divided by number of observational min, was subtracted from the previous value to obtain the overall aggression score. Sample sizes were as follows: chase = 9,control and sham = 7, chase + 1 and cortisol = 6, metyrapone = 5.
% Time on territory Average # of digs S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 Table 3Pink salmon (Oncorhynchus gorbuscha) behavior profiles for ripe fish during 4 day trials; values are stated as mean (±SE). All data were analyzed using Wilcoxon Rank-Sum testsand Tukey's HSD test was used to determine where differences lay when a significant effect was obtained (noted by letter scores). All data that are expressed as percentages weretransformed into ArcSine (square root) values before being analyzed. Data for all variables except the aggression score were averaged over days that fish were on established ter-ritories. Aggression scores were added for all days spent on territories and divided by number of observational min. Each fish had a similar score for aggressive attacks against, andthis score, divided by number of observational min was subtracted from the previous value to obtain the overall aggression score. Sample sizes were as follows: metyrapone = 10,sham, cortisol, control and chase + 1 = 9 and chase = 7.
% Time on territory Average # of digs at the Weaver Creek spawning channel, related between the effects of baseline cortisol and stress-induced cortisol on mortality to changes in physiological condition and activity levels, providing a baseline of variables that change as Pacific salmon (spe- There was evidence that metyrapone treatment caused some egg cifically sockeye salmon) senesce. To complement that work, the pre- retention and delayed senescence, as observed in the enclosure sent study attempted to identify whether stressful conditions can study (i.e., significantly lower cortisol values compared with other cause pre-spawn mortality on spawning grounds. It seems plausible treatment groups in ripe fish). If cortisol spikes immediately prior to that since cortisol treatment in the present study increased cortisol ovulation ), this process could have been inhibited values to those found in senescing fish and at the same time reduced through the action of metyrapone in blocking cortisol synthesis. Addi- longevity, then the premature mortality we observed was a function tionally, cortisol rises again during senescence (), of this senescence-like physiological state, a state that was not and this process also could have been inhibited by the action of reached via the imposition of acute stressors, even though exposure metyrapone. To examine these possibilities, more detailed time to acute stressors was expected to acutely elevate circulating cortisol course of plasma hormone levels is needed. Ideally, metyrapone- treated fish should be monitored just prior to ovulation, immediatelyfollowing egg release and before morbidity.
Elevated cortisol levels on spawning grounds Unripe cortisol-treated fish spent less time on their territory than all other groups. In addition, cortisol-treated fish were significantly One of the most notable findings of this study was that exposure of less aggressive than fish in the other treatment groups, and were fre- pink salmon to acute stressors on spawning grounds did not alter quently subjected to aggressive attacks from conspecifics. A decrease spawning ground longevity, reproductive success, or behavior, in ac- in aggressiveness is detrimental to reproductive success because a fe- cordance with theory that semelparous animals in general should resist male benefits from guarding its territory from other females looking stress (i.e. attenuate stress responses and/or exhibit resistance to the for suitable habitat, and aggressive behavior is often associated with effects of elevated stress hormone levels) in favor of allocating energy reproductive success (). These re- to their current, and only, reproductive opportunity sults are supported by previous studies that found that cortisol treat- ). Behavioral and physiological profiles of spawning ment increased the probability of individual fish (rainbow trout in Pacific salmon are well documented, but the function of (baseline) cor- these cases) experiencing increased fin damage indicative of both ag- tisol elevation in semelparous fish in their natural spawning habitat is gressive attacks (and becoming socially not well understood. From a mechanistic standpoint, it has yet to be subordinate (No behav- determined how semelparous salmon successfully breed despite circu- ioral differences were detected among treatments for ripe fish. This lating cortisol being elevated to a level that would inhibit reproduction finding suggests that even in the face of chronically elevated cortisol in other species. However, our data indicate that there is a limit to this levels, reproductively mature fish maintain key reproductive behav- capacity because cortisol treatment did impair reproduction.
iors, further supporting the idea that fish with limited reproductive The scope of the present study does not enable us to speculate opportunity will still engage in spawning in what would be regarded about the mechanism of cortisol elevation on spawning grounds. More- as extreme situations during other life-history phases.
over, we did not measure cortisol receptor occupancy or sensitivity,factors that will affect the ability of (high) cortisol levels to mediate Metyrapone treatment target tissue responses, and an issue that ideally would be addressedin future studies. We can conclude, however, that acute elevation of Metyrapone inhibits the enzyme 11-β hydroxylase, thereby cortisol levels does not hinder reproductive behaviors and outcome.
preventing synthesis of cortisol from 11-deoxycortisol In addition, it seems that the second spike in cortisol is an indicator No significant changes in cortisol titers, reproductive behavior, re- of impending senescence, as noted in previous studies (e.g., productive success, or hormone levels occurred as a result of metyrapone ). If high cortisol levels are evident before spawning is com- treatment. determined that metyrapone inhibits the plete, key reproductive behaviors and outcome can be negatively af- cortisol response to a stressor but does not reduce baseline (non-stressed) fected, as evidenced in this study by the use of semi-chronic cortisol cortisol levels. There is also a suggestion that plasma cortisol does not turn implants. It would have been useful to collect blood immediately fol- over rapidly for semelparous salmon on spawning grounds lowing exposure of fish to the acute stressors to assess the extent of Therefore, it is possible that baseline (i.e. non- the stress response elicited. In a similar study on stress responsiveness, stressed) levels of cortisol were maintained, but increases in cortisol levels observed an increase in cortisol levels from 333 ± 17 with stress were prevented (although this was not tested in the current to 497 ± 22 ng ml−1 following 2 min of air exposure using Weaver study). For future studies, responsiveness could be observed following in- Creek sockeye salmon. Other Pacific salmonids (including sockeye, jection to determine whether metyrapone-treated fish respond to acute chum [O. keta], coho [O. kisutch] and Chinook [O. tshawtscha]), as well stressors. This approach would provide a useful means of distinguishing as pink salmon, all have been found to experience an acute stress S.H. McConnachie et al. / Hormones and Behavior 62 (2012) 67–76 response when exposed to short-term stressors, with cortisol levels re- covering within 2–4 h (Mike Donaldson, UBC, personal communica-tion). Therefore, the pink salmon in this study likely experienced an Barry, T.P., Marwah, A., Nunez, S., 2010. Inhibition of cortisol metabolism by 17α, 20β-P: mechanism mediating semelparity in salmon? Gen. Comp. Endocrinol.
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Personalised, Serendipitous and Diverse Linked Data Resource Recommendations Milan Dojchinovski and Tomas Vitvar Web Intelligence Research Group Faculty of Information Technology Czech Technical University in Prague Abstract. Due to the huge and diverse amount of information, the ac-tual access to a piece of information in the Linked Open Data (LOD)cloud still demands significant amount of effort. To overcome this prob-lem, number of Linked Data based recommender systems have been de-veloped. However, they have been primarily developed for a particulardomain, they require human intervention in the dataset pre-processingstep, and they can be hardly adopted to new datasets. In this paper, wepresent our method for personalised access to Linked Data, in particularfocusing on its applicability and its salient features.

Allergology international vol.56 no.

Allergology International. 2007;56:37-43DOI: 10.2332! Awarded Article, Annual Meeting of JSA The Relationship between ExhaledNitric Oxide Measured with an Off-lineMethod and Airway ReversibleObstruction in Japanese Adults withAsthmaTakahiro Tsuburai1, Naomi Tsurikisawa1, Masami Taniguchi1, Sonoko Morita1, Emiko Ono1,Chiyako Oshikata1, Mamoru Ohtomo1, Yuji Maeda1, Kunihiko Ikehara2 and Kazuo Akiyama1