Temporal changes in blood variables during ﬁnal maturation...

Journal of Fish Biology (2011) 79, 449–465

doi:10.1111/j.1095-8649.2011.03042.x, available online at wileyonlinelibrary.com

Temporal changes in blood variables during finalmaturation and senescence in male sockeye salmon

Oncorhynchus nerka: reduced osmoregulatory ability canpredict mortality

K. M. Jeffries*†, S. G. Hinch*‡, M. R. Donaldson*, M. K. Gale*,J. M. Burt*, L. A. Thompson§, A. P. Farrell‖, D. A. Patterson§ and

K. M. Miller¶

*Centre for Applied Conservation Research and Department of Forest Sciences, University ofBritish Columbia, 2424 Main Mall, Vancouver, British Columbia, V6T 1Z4 Canada, ‡Institute

for Resources, Environment and Sustainability, University of British Columbia, 2202 MainMall, Vancouver, British Columbia, V6T 1Z4 Canada, §Fisheries and Oceans Canada,

Cooperative Resource Management Institute, Simon Fraser University, 8888 University Drive,Burnaby, British Columbia, V5A 1S6 Canada, ‖Land and Food Systems and Department of

Zoology, University of British Columbia, 2357 Main Mall, Vancouver, British Columbia, V6T1Z4 Canada and ¶Fisheries and Oceans Canada, Molecular Genetics Section, Pacific

Biological Station, 3190 Hammond Bay Road, Nanaimo, British Columbia, V9T 6N7 Canada

(Received 23 June 2010, Accepted 13 May 2011)

This study is the first to characterize temporal changes in blood chemistry of individuals fromone population of male sockeye salmon Oncorhynchus nerka during the final 6 weeks of sexualmaturation and senescence in the freshwater stage of their spawning migration. Fish that diedbefore the start of their historic mean spawning period (c. 5 November) were characterized by a20–40% decrease in plasma osmolality, chloride and sodium, probably representing a complete lossof osmoregulatory ability. As fish became moribund, they were further characterized by elevatedlevels of plasma cortisol, lactate and potassium. Regressions between time to death and plasmachloride (8 October: P < 0·001; 15 October: P < 0·001) indicate that plasma chloride was a strongpredictor of longevity in O. nerka. That major plasma ion levels started to decline 2–10 days (meanof 6 days) before fish became moribund, and before other stress, metabolic or reproductive hormonevariables started to change, suggests that a dysfunctional osmoregulatory system may initiate rapidsenescence and influence other physiological changes (i.e. elevated stress and collapsed reproductivehormones) which occur as O. nerka die on spawning grounds. © 2011 The Authors

Journal of Fish Biology © 2011 The Fisheries Society of the British Isles

Key words: osmolality; osmoregulation; Pacific salmon; plasma chloride; prespawn mortality;spawning migration.

INTRODUCTION

When adult sockeye salmon Oncorhynchus nerka (Walbaum 1792) migrate upriver totheir natal spawning area, they have already ceased feeding, are rapidly maturing andare undergoing dramatic morphological changes in preparation for a single spawning

†Author to whom correspondence should be addressed. Tel.: +1 604 822 1969; email: [email protected]

449© 2011 The AuthorsJournal of Fish Biology © 2011 The Fisheries Society of the British Isles

450 K . M . J E F F R I E S E T A L .

event. Because O. nerka are semelparous, they die shortly after spawning. The rapidsenescence that occurs postspawning in adult Pacific salmon Oncorhynchus spp. ischaracterized by immunosuppression and organ deterioration (Dickhoff, 1989; Finch,1990). If however the fishes die before spawning, either in river (termed en routemortality) or on the spawning grounds (termed prespawn mortality), lifetime fitnessis zero. Prespawn mortality rates are highly variable but can reach 90% in someyears (mean rates of 3·3–23·7%) (Gilhousen, 1990). There are many factors thatpotentially contribute to these mortalities, e.g. high water temperature (Crossin et al.,2008; Farrell et al., 2008), high discharge (Rand et al., 2006), parasites and disease(Gilhousen, 1990; Jones et al., 2003), and possibly increased rates of senescence, butat present there are no reliable indicators to predict whether an individual arrivingat a spawning area will in fact survive to spawn.

Rapid biosampling (Cooke et al., 2005) offers a snapshot of the physiologicalstatus of migrating Oncorhynchus spp., which includes characteristics associatedwith en route mortality (Cooke et al., 2006; Crossin et al., 2009). There is a decreasein plasma osmolality and chloride levels as adult O. nerka migrate in fresh water(Shrimpton et al., 2005), which suggests a progressively reduced osmoregulatoryability during freshwater spawning migrations. There also appears to be an increase inindices of stress during spawning ground residence (Hruska et al., 2010). The presentstudy tested the hypothesis that individual fish would show signs of physiologicalimpairment as they senesce. It was predicted that fish nearing death would show signsof reduced osmoregulatory ability, have elevated indices of stress and decreased sexsteroid levels when compared with fish that survived to the mean historic spawningperiod. To test this hypothesis, the same individuals were repeatedly sampled forblood to characterize changes in blood chemistry associated with mortality and toquantify temporal patterns in blood chemistry during final maturation and senescencein adult male O. nerka.

MATERIALS AND METHODS

F I S H C O L L E C T I O N A N D H A N D L I N G

Male O. nerka from the Harrison Rapids population were collected from the HarrisonRiver, British Columbia, which is a major tributary of the Fraser River. Seventy-eight fishwere collected on Chehalis First Nation’s land downstream of Harrison Lake by beach seinefrom 15 to 18 September 2008 (Fig. 1). Harrison Rapids O. nerka may enter the Fraser River6–11 weeks prior to spawning in the Harrison River (Lapointe, 2009) and hold in the Harri-son River or in Harrison Lake until spawning immediately downstream of the collection site.Fish were transported live to the Fisheries and Oceans Canada Cultus Lake Laboratory (c.45 min by vehicle) and upon arrival, were PIT-tagged and an adipose fin clip was taken forDNA stock identification (Beacham et al., 2005). The PIT-tagged fish were held in a large c.20 000 l stock tank at c. 10·5◦ C. Harrison River water temperature during collection rangedbetween 14 and 18◦ C. Eleven of the fish collected, used as controls for potential effectsof repeated handling, were not PIT-tagged or immediately sampled for DNA and placed inseparate tanks after being transported to the Cultus Lake Laboratory. On 22–23 September2008, fish were removed by dip-net from the large stock tank and immediately sampled forblood. Fish were then placed into smaller c. 8000 l tanks for ease of repeat sampling. Thesame individual fish were re-sampled on 8 October, 15 October and 5 November 2008. Fishthat no longer maintained equilibrium, but were still ventilating, were terminally sampledthroughout the experiment and designated ‘moribund’. Little is known about the migration

© 2011 The AuthorsJournal of Fish Biology © 2011 The Fisheries Society of the British Isles, Journal of Fish Biology 2011, 79, 449–465

B L O O D VA R I A B L E S D U R I N G S E N E S C E N C E I N O . N E R K A 451

BritishColumbia

Vancouver

FraserRiver

HarrisonRiver

HarrisonRiver collectionsite

HarrisonLake

Weaver Creekspawningchannel

10 km

Cultus LakelaboratoryCanada

U.S.A.

N

Fig. 1. Map of the lower Fraser River, British Columbia, Canada, with the locations of the Harrison Rivercollection site and Cultus Lake Laboratory.

behaviour of Harrison Rapids O. nerka, but co-migrating Weaver Creek O. nerka, whichspawn in a tributary of the Harrison River near the capture site (Fig. 1), are known to migrateinto and occupy the cool hypolimnion of nearby Harrison Lake (c. 6 km upstream of capturesite). Telemetry tracking studies have revealed that the use of cool water as a thermal refugeenhances chances of survival to spawning, especially for fish that encounter high river tem-peratures during migration (Farrell et al., 2008; Mathes et al., 2010). To mimic fish utilizinga thermal refuge, such as those available in Harrison Lake, water temperature was decreasedto c. 7◦ C on 5 October. On 5 November 2008, which is approximately the beginning ofthe natural historic spawning period for Harrison Rapids O. nerka (peak spawning occurredbetween 11 and 13 November 2008), all fish, including control fish, were terminally sam-pled. Terminal sampling included a blood sample, as well as measurement of lengths (LPOH,postorbital–hypural bone length), body mass (MB; wet mass, ±10 g), body depth (DB), liver(ML) and gonad mass (MG; wet mass, ±1 g), and kype length (LKype). Kype can increase inlength throughout sexual maturation in male O. nerka and is considered a secondary sexualcharacteristic (Hendry & Berg, 1999).

Fish were categorized into groups based on longevity during the holding period (Table I).Group 1 died within a week of 8 October, group 2 died after two rounds of sampling between8 and 15 October, group 3 died after three rounds of sampling between 15 October and5 November and group 4 survived until 5 November, when they were terminally sampled.


452 K . M . J E F F R I E S E T A L .

Tab

leI.

Mea

n±

s.d.

post

orbi

tal–

hypu

ral

bone

leng

th(L

PO

H),

wet

body

mas

s(M

B),

body

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h(D

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kype

leng

th(L

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e),

wet

liver

mas

s(M

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and

wet

gona

dm

ass

(MG)

and

sam

ple

size

s(n

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mor

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ree

mor

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,su

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and

cont

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Onc

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rka

Sam

ple

grou

pn

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OH

(cm

)*M

B(g

)*D

B(c

m)

LK

ype

(cm

)M

L(g

)**

MG

(g)

Die

dbe

fore

8O

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er13

52·04

±1·3

0a30

12·31

±32

5·17a

14·38

±0·9

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6±

0·71a

67·55

±10

·53a

90·16

±24

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ter

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1352

·36±

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3115

·85±

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7·57

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14·97

a96

·18±

32·71

ab

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thre

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ds18

53·04

±3·3

8a30

09·22

±45

3·44a

14·78

±1·0

2a7·6

2±

0·75a

70·26

±14

·93a

76·08

±21

·53a

Surv

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53·55

±2·6

0a33

12·91

±57

3·50a

16·64

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8b8·2

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1·17a

71·30

±6·5

7b89

·94±

10·42

ab

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trol

s9

50·70

±2·1

0a30

64·63

±36

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16·44

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65·97

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0·48

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Only fish that survived a minimum of 2 days after being sampled were included in thesurvival group comparisons to minimize the potential for handling to have contributed to thesubsequent mortality. Fish were only included in the survival group analysis if there was amoribund or terminal blood sample matched with other live samples for an individual fish.All fish were used to calculate regressions between time to death and plasma chloride.

B L O O D S A M P L I N G

Blood samples (c. 3 ml) were taken from fish by caudal puncture using a 21 gauge needleand a heparinized vacutainer. Whole-blood samples were centrifuged for 6–7 min and plasmasamples were placed into three 0·5 ml tubes for storage at −86◦ C until subsequent analysis.Plasma osmolality, ions (sodium, chloride, potassium), glucose and lactate were measuredusing the procedures outlined in Farrell et al. (2001). Plasma cortisol, testosterone and 17β-oestradiol were measured using commercial enzyme-linked immunosorbent assay (ELISA)kits (Neogen Corporation; www.neogen.com). Testosterone and 17β-oestradiol samples wereextracted in ethyl ether according to manufacturer’s protocols. Cortisol, testosterone and17β-oestradiol samples were all run in duplicate at appropriate dilutions.

S TAT I S T I C A L A NA LY S I S

All statistical tests were performed using SAS software version 9.1 (SAS Institute; www.sas.com). ANOVA was used to test for differences between groups for MB, LPOH and LKype.In all cases, homogeneity of variances was assessed by Bartlett’s test and normality wastested using Kolmogorov–Smirnov tests (Sokal & Rohlf, 1995). Data were log10-transformedif the assumption of homogeneity of variances could not be met. Where the assumptionsof normality could not be met (DB only), the non-parametric Kruskal–Wallis test was used(Sokal & Rohlf, 1995). Mass and length relationships between groups were compared usingANCOVA with LPOH as the covariate. Relative ML and relative MG (as a percentage of MB)were compared using ANCOVA with adjusted MB (MB − MOrgan) as the covariate.

Plasma variables were analysed using a repeated measures split-plot ANOVA, with thesample time within a group treated as the subplot and sample groups as the whole plot.Multiple pair-wise comparisons were made using the Bonferroni adjustment method (Zar,1999). There were 26 possible comparisons (within groups and between groups at a singlesample time), which made a highly conservative critical α = 0·0019 after the Bonferroni cor-rection. When appropriate, significant differences before Bonferroni correction are discussed.Samples taken from fish that became moribund were only compared within their group.Comparisons between the survivors and the control fish on the day that the experiment wasterminated (5 November 2008) were assessed using t-tests. Data were log10-transformed if theassumption of homogeneity of variances was not met. If the assumption of normality couldnot be met, non-parametric Wilcoxon tests were used (Sokal & Rohlf, 1995). Relationshipsbetween plasma chloride levels and time to death post-sampling for each sampling date wereassessed using linear regressions on log10-transformed data.

RESULTS

M O R P H O L O G Y

Among survival groups, there were no differences between LPOH (ANOVA, d.f. =4,58, P > 0·05), MB (ANOVA, d.f. = 4,59, P > 0·05) and LKype (ANOVA, d.f. =4,59, P > 0·05) (Table I). When comparing mass and length relationships betweentreatment groups, the individuals from the control group were heavier for a givenLPOH than group 2 individuals (ANCOVA, d.f. = 4,57, P < 0·001). Individuals from


454 K . M . J E F F R I E S E T A L .

group 4 (survivors) and control groups had greater DB than individuals that diedbefore 5 November (Kruskal–Wallis, d.f. = 4, P < 0·001). The slopes of the MLto adjusted MB relationships were different between individuals from the survivorsand the group 2 individuals (ANCOVA, d.f. = 4,58, P < 0·05). Individuals from thecontrol group had heavier MG for a given MB than group 3 individuals (ANCOVA,d.f. = 4,58, P < 0·001).

B L O O D VA R I A B L E S

There was an overall decrease in plasma osmolality (split-plot ANOVA, withingroup: d.f. = 3,51, P < 0·001; between sampling times: d.f. = 3,104, P < 0·001;group–time interaction: d.f. = 6,104, P < 0·001), chloride ions (split-plot ANOVA,within group: P < 0·001; between sampling times: P < 0·001; group–time inter-action: P < 0·001) and sodium ions (split-plot ANOVA, within group: P < 0·001;between sampling times: P < 0·001; group–time interaction: P < 0·001) with val-ues decreasing sharply in moribund fish (Fig. 2). The difference in osmolality on15 October between group 4 (survivors) and group 3 was significant at P < 0·05,but not after Bonferroni correction [Fig. 2(a)]. Plasma chloride levels were signif-icantly lower before a fish died when compared to group 4 (survivors) and werethe best predictor of survival [Fig. 2(b)]. The difference in sodium ion means on15 October between the group 4 (survivors) and group 3 individuals was significantat the P < 0·05 level, but not after Bonferroni correction [Fig. 2(c)]. For all threeblood plasma variables, there were no significant differences between the group 4(survivors) and the control fish on 5 November. Because two of the 11 control fishdied during the holding period, there were only nine control fish that were terminallysampled on 5 November.

Because chloride was the best predictor of longevity according to the split-plotANOVAs, only regressions between chloride and number of days until death are pre-sented. There was a significant relationship between plasma chloride levels and timeto death (Fig. 3) for samples taken on 8 October (regression, d.f. = 48, P < 0·001,r2 = 0·830) and 15 October (regression, d.f. = 29, P < 0·001, r2 = 0·751). Therewas no significant relationship, however, between plasma chloride levels and timeto death for samples taken on 22–23 September (regression, d.f. = 65, P > 0·05,r2 = 0·009).

Within a sampling group, 17β-oestradiol levels decreased when compared to ini-tial samples taken from 22 to 23 September, and moribund fish had the lowest levelsof 17β-oestradiol [Fig. 4(a); split-plot ANOVA; within group: P < 0·01; betweensampling times: P < 0·001; group–time interaction: P < 0·01]. Testosterone levelsgenerally increased with time within a sampling group compared to initial sam-ples and decreased in moribund fish [Fig. 4(b); split-plot ANOVA; within group:P < 0·01; between sampling times: P < 0·001; group–time interaction: P < 0·01].For both hormones, there were no significant differences between group 4 (survivors)and control fish at the termination of the experiment.

Cortisol levels decreased over time, but increased dramatically in moribund fish[Fig. 5(a); split-plot ANOVA, within group: P < 0·001; between sampling times:P < 0·001; group–time interaction: P < 0·001]. Conversely, glucose levels and thevariability in the glucose values generally increased with time [Fig. 5(b); split-plotANOVA, within group: P < 0·01; between sampling times: P < 0·001; group–time



320(a) (b)

140

130

120

110

100

90

80

70

60

50

310

300

290

M +

+

++

+

++ +

+++

MM

M

MM

280

Plas

ma

osm

olal

ity (

mO

sm k

g–1)

Plas

ma

chlo

ride

(m

mol

l–1)

270

260

250

240

2300 5 10 15 20 25 30 35 40 45 50

Experiment day Experiment day

0 5 10 15 20 25 30 35 40 45 50

(c)

+

+

+ +

+

M

MM

Plas

ma

sodi

um (

mm

ol l–1

)

160

150

140

130

120

110

100

900 5 10 15 20

Experiment day

25 30 35 40 45 50

Fig. 2. Plasma levels of (a) osmolality, (b) chloride and (c) sodium sampled from male Harrison RapidsOncorhynchus nerka over the final 6 weeks of maturation and senescence in 2008 [ , died before8 October (group 1); , died after two rounds (group 2); , died after three rounds (group 3); ,survivors (group 4); , controls]. Data are presented as means ± s.d. Statistical differences from the 22to 23 September 2008 (experiment day 1) sampling date within groups are indicated (+) and differencesbetween groups and the survivors group at a given time are indicated (∗). The 8 October, 15 October and5 November 2008 samplings occurred on experiment day 16, 23 and 44, respectively. Mean experimentday and value of samples taken including raw data (smaller data points) as fish became moribund areindicated (M). Control fish were sampled on experiment day 44.

interaction: P < 0·001]. Plasma lactate, like cortisol, increased dramatically in mori-bund fish [Fig. 5(c); split-plot ANOVA, within group: P < 0·001; between samplingtimes: P < 0·001; group–time interaction: P < 0·001]. Plasma potassium levelsincreased in moribund fish [Fig. 5(d)], however, the increase was not always sig-nificant within the sample groups (split-plot ANOVA, within group: P < 0·001;between sampling times: P < 0·001; group–time interaction: P < 0·001). For thesefour blood plasma variables, there were no significant differences between group 4(survivors) and the control fish on 5 November.


456 K . M . J E F F R I E S E T A L .

45N

umbe

r of

day

s un

til d

eath

40

35

30

25

20

15

10

5

070 75 80 85 90 95 100

Plasma chloride (mmol l–1)

105 110 115 120 125 130 135 140 145

Fig. 3. Relationship between the number of days to death (post-sampling) and plasma chloride levels in maleHarrison Rapids Oncorhynchus nerka sampled on 22–23 September ( ), 8 October ( , ) and 15October ( , ) 2008. The curves were derived from the linear regression estimates of the slope andintercept for log10-transformed data to fit a power function and are 8 October y = 4·09E −12x5·99 and15 October y = 2·04E − 12x6·17.

DISCUSSION

Depletion of energy reserves, physiological stress caused by metabolic or dis-ease issues and impaired osmoregulatory functions are all proposed mechanisms toaccount for the rapid senescence and mortality of Oncorhynchus spp. at the spawningground (Shrimpton et al., 2005; Hruska et al., 2010). Understanding the mechanismsof the rapid senescence has been hampered by the fact that there have been no studieson wild Oncorhynchus spp. that quantify temporal changes in blood chemistry ofindividuals in one population during their final weeks of life. The present study foundthat plasma osmolality, chloride and sodium levels decreased during the final weeksof life in adult male O. nerka, followed by a further decrease (c. 20–40%) as fishbecame moribund. This final decrease in plasma ions probably represents a completeloss of osmoregulatory ability. The relationship between plasma chloride levels andtime to death became stronger as fish approached the historic peak spawning period,which suggests that the ability of plasma ion levels to predict mortality is proba-bly more applicable to O. nerka on or approaching the spawning grounds. Majorplasma ion levels started to decline 2–10 days (mean of 6) before fish became mori-bund, and before other stress, metabolic or reproductive variables started to change,which suggests that osmoregulatory dysfunction may represent the initiation of rapid



0·18

0·16

0·14

0·12

0·10

0·08

0·06

Plas

ma

17 β

-oes

trad

iol (

ng m

l–1)

Plas

ma

test

oste

rone

(ng

ml–1

)

0·04

0·02

0·00

35

30

25

20

15

10

5

0

–5

(b)

(a)

0 5

M

M

M

M

M

M

++

+

+

+

+

+

+

10 15 20 25

Experiment day

30 35 40 45 50

Fig. 4. Plasma levels of (a) 17β-oestradiol and (b) testosterone sampled from male Harrison RapidsOncorhynchus nerka over the final 6 weeks of maturation and senescence in 2008 [ , died before8 October (group 1); , died after two rounds (group 2); , died after three rounds (group 3); ,survivors (group 4); , controls]. Data are presented as means ± s.d. Statistical differences from the 22to 23 September 2008 (experiment day 1) sampling date within groups are indicated (+). One samplefrom the 8 October sample from the after three rounds of sampling group was below the detection limitsfor the testosterone assay and therefore was not included in the mean calculation. The 8 October, 15October and 5 November 2008 samplings occurred on experiment day 16, 23 and 44, respectively. Meanexperiment day and value of samples taken as fish that became moribund are indicated (M). Control fishwere sampled on experiment day 44.


458 K . M . J E F F R I E S E T A L .

Experiment day

28

24

20

16

12

6

4

2

0

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ma

cort

isol

(ng

ml–1

)Pl

asm

a la

ctat

e (m

mol

l–1)

1200(a) (b)

(c) (d)

1000

800

600400

200

0

MM

M++

+ +

+

+

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M

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+

+

0 5 10 15 20 25 30 35 40 45 50

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ma

gluc

ose

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ol l–1

)Pl

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a po

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mol

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Fig. 5. Plasma levels of (a) cortisol, (b) glucose, (c) lactate and (d) potassium sampled from male HarrisonRapids Oncorhynchus nerka over the final 6 weeks of maturation and senescence in 2008 [ , diedbefore 8 October (group 1); , died after two rounds (group 2); , died after three rounds (group 3);

, survivors (group 4); , controls]. Data are presented as means ± s.d. Statistical differences fromthe 22 to 23 September 2008 (experiment day 1) sampling date within groups are indicated (+) anddifferences between groups and the survivors group at a given time are indicated (∗). The 8 October, 15October and 5 November 2008 samplings occurred on experiment day 16, 23 and 44, respectively. Meanexperiment day and value of samples taken as fish that became moribund are indicated (M). Control fishwere sampled on experiment day 44.

senescence. Osmoregulatory dysfunction may be a precursor to other physiologicalchanges (i.e. elevated stress and collapsed reproductive hormones) that the presentstudy and others have found in moribund O. nerka (Hruska et al., 2010).

The causes of a precursory reduction in major plasma ions are unclear but mayinvolve an inability to resorb ions at the kidney, a sequestering of ions in thebody, a loss of ions across body surfaces and an inability to take ions up at thegill. Because immune functions become impaired in O. nerka on the spawningground (Miller et al., 2009), naturally occurring diseases, particularly those whichaffect kidney or gill function (Wagner et al., 2005; Bradford et al., 2010a), maybe partially responsible for such osmoregulatory problems. Reductions in plasmaions as fishes approach historic spawning periods also appear to occur in adult coho



salmon Oncorhynchus kisutch (Walbaum 1792) (Donaldson et al., 2010) and Chinooksalmon Oncorhynchus tshawytscha (Walbaum 1792) (T. D. Clark, unpubl. data),which suggests that osmoregulatory dysfunction may be a common phenomenon inall senescing semelparous Oncorhynchus spp.

Finch (1990) hypothesized that rapid senescence in adult Oncorhynchus spp.postspawning may be related to elevated cortisol levels that lead to the deteriora-tion of the kidney, liver and spleen and to overall immunosuppression. The observeddecline in plasma cortisol levels over the 6 week interval of the present study has beenobserved previously (Fagerlund, 1967; Hinch et al., 2006). The decline was followedby a rapid four to nine-fold increase as fish became moribund. Such an increase maybe caused by decreased cortisol clearance by the kidney and liver and is potentiallyrelated to organ degeneration in Oncorhynchus spp. (Finch, 1990; Carruth et al.,2000). Elevated cortisol levels have also been detected in diseased O. nerka daysbefore they died, possibly as a response to fungal or bacterial infections (Fagerlund,1967). As fish in the present study were not treated with antibiotics, a disease-induced increase in cortisol levels is plausible, as increased incidence of disease iscommonly observed in Oncorhynchus spp. held in captivity. Additionally, fishes maybecome immunocompromised when stressed (Schreck et al., 2001); these stressorsmay include confinement and sampling stress. An impaired immune response dueto sampling and confinement stress may have made some of the sampled fish moresusceptible to disease, fungal and parasite infection and contributed to the elevatedcortisol levels near death and the overall increased mortality in the sampled groupcompared to the control group. Both direction and magnitude of the increases inplasma cortisol levels in the present study, however, were similar to those observedin moribund O. nerka from spawning grounds at Weaver Creek, a population whichspawns near the Harrison Rapids population (Fig. 1; Hruska et al., 2010). Therefore,it is difficult to distinguish between the possibility that the elevated cortisol lev-els are associated with general disease progression and immunosuppression, whichcommonly occurs in senescing fishes, v. senescence alone. Regardless, it can beconcluded that elevated cortisol is characteristic of mortality in Oncorhynchus spp.

Cortisol plays a large role in osmoregulation and metabolism in fishes as it hasboth mineralcorticoid and glucocorticoid roles (Mommsen et al., 1999). Mineral-corticoids function mainly in the regulation of hydromineral balance (Milla et al.,2009). Shrimpton et al. (2005) showed that Na+,K+-ATPase activity, which may beregulated by cortisol, increases in spawning O. nerka and that the increased Na+,K+-ATPase activity on the spawning grounds is an attempt to compensate for an osmoticperturbation during spawning. Degeneration of the kidney due to prolonged elevationof cortisol and resulting loss of kidney function could result in the dramatic loss ofosmoregulatory ability detected in the present study. Previous work has shown thatsevere parasite-induced kidney damage in adult O. nerka is correlated with a decreasein plasma osmolality, indicative of reduced osmoregulatory ability (Bradford et al.,2010b).

There was an increase in plasma glucose levels during the final 6 weeks ofmaturation and senescence. Cortisol has a metabolic role in fishes, which includesenhancing the rate of gluconeogenesis in the liver (Mommsen et al., 1999). Althoughthe evidence is not always consistent, an increase in cortisol may eventually result inelevated plasma glucose levels in fishes (Mommsen et al., 1999). In male O. nerkacaught throughout the reproductive season, elevated plasma glucose levels generally


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occurred when cortisol levels were also high (Kubokawa et al., 1999). The increasein plasma glucose levels in the present study did not necessarily correspond to ele-vated cortisol levels. Glucose levels often increase when a fish is confined (Portzet al., 2006). Plasma glucose, however, is known to increase during the period offinal maturation in O. nerka (Kubokawa et al., 1999). Patterson et al. (2004) foundno difference in glucose levels between O. nerka captured during migration andheld in captivity and fish from the same population collected from natal spawninggrounds. Therefore, the general increase in plasma glucose levels in the present studymay be due to natural processes associated with final maturation and senescence,rather than a response to confinement stress. Furthermore, there was higher among-individual variability in plasma glucose during later sampling periods, as fish becamemore mature, suggesting that there may be multiple physiological mechanisms thataffect individual plasma glucose levels during final maturation and senescence inOncorhynchus spp., an interpretation also supported by other research on O. nerkaat spawning grounds (Hruska et al., 2010).

Blood-circulating sex steroids, such as testosterone and 17β-oestradiol, increaseduring reproductive maturation and peak in males well before spawning (some-times several hundred kilometres before reaching natal spawning grounds and severalweeks before final maturation), then diminish and continue to decline prior to deathin O. nerka (Kubokawa et al., 1999; Hinch et al., 2006), pink salmon Oncorhynchusgorbuscha (Walbaum 1792) (Dye et al., 1986; Williams et al., 1986), O. kisutch(Fitzpatrick et al., 1986) and chum salmon Oncorhynchus keta (Walbaum 1792)(Onuma et al., 2009). Despite an extended holding period that should have spannedthe timing of previously observed peaks in sex steroids, there was only a moderateincrease in testosterone during the experiment, and no increase in 17β-oestradiol.Acute confinement stress (up to 30 min) has been shown to reduce blood-circulatingsex steroid levels in O. nerka (Kubokawa et al., 1999), probably due to negativefeedback control mechanisms by the hypothalamic–pituitary–interrenal axis duringperiods of stress (Wendelaar Bonga, 1997). While it is possible that confinementstress may have been a factor in the present study, Patterson et al. (2004) found thatsex steroid levels in O. nerka held for 4 weeks were comparable to levels detectedin fish collected from natal spawning sites, which indicates captivity may not neces-sarily result in reduced blood-circulating sex steroid levels. Alternatively, the peakin 17β-oestradiol and testosterone may have already occurred in the Harrison Rapidspopulation before the September collection date. Elevated cortisol, as observed in fishthat became moribund in the present study, has been shown to inhibit the reproduc-tive cycle in male fish and has been associated with reductions in blood-circulatingsex steroid levels in migrating O. nerka during periods of acute stress (Hinch et al.,2006) and in postspawned death in O. nerka held in captivity (Kubokawa et al.,1999). The low levels of sex steroids detected near death in the present study maybe associated with these dramatically increased plasma cortisol levels that occuras O. nerka become moribund. Decreases in 17β-oestradiol and testosterone levelsoccurred earlier in fish that did not survive to the mean historic spawning period,which may suggest a disconnect between maturation and mean spawning time in fishthat suffer prespawn mortality. Because fish that died prematurely had smaller MGand DB (a secondary sexual characteristic), it is probable that these fish died beforethey became fully mature, in contrast to the MG and DB in fish in the survivors andcontrol groups. It is unclear whether senescence occurs because of reproductive state



or whether the advanced reproductive state is because the fish are further along asenescence trajectory.

Plasma lactate and potassium increased dramatically in fish that became moribund.Plasma lactate, a by-product of anaerobic metabolism, increases post-exercise andmay increase post-stress in fishes (Barton et al., 2002). Following periods of exercise,potassium levels increase in blood plasma due to a loss of potassium from musclecells (Sejersted & Sjogaard, 2000). Plasma potassium can also increase during peri-ods of hypoxia, possibly indicative of cell damage or muscle depolarization (Mateyet al., 2008). In rainbow trout Oncorhynchus mykiss (Walbaum 1792) hepatocytes,potassium effluxes accompany cellular apoptosis and may be a factor in initiatingapoptosis (Krumschnabel et al., 2007). Therefore, the increase in plasma potassiumlevels and lactate is probably indicative of activity, stress and cellular degradation,which may be associated with final senescence in Oncorhynchus spp.

Sampling and confinement stress could potentially have influenced the patternsin blood properties in the sampled fish. Adult Oncorhynchus spp. exhibit tran-sient increases in plasma cortisol, glucose and lactate after a single stress event,which then return to pre-stressor levels (Donaldson et al., 2010). When exposed to achronic stressor, these plasma variables may return to baseline values after a periodof acclimation (Trenzado et al., 2003; Cairns et al., 2008). Because the magnitudeand direction of change in the blood variables was consistent with previous stud-ies on spawning ground fish (Hruska et al., 2010) and with control fish that werenot previously sampled, the patterns described in the present study are probablycharacteristic of the final weeks of final maturation and senescence in O. nerka.

In conclusion, the present study characterized the temporal changes in blood prop-erties and documented the initial osmoregulatory failure of male O. nerka duringthe freshwater stage of their spawning migration, and subsequent elevated stressand depressed reproductive hormone levels which characterize final maturation andsenescence. Because rapid declines in osmoregulatory ability preceded death by2–10 days, specific levels of major plasma ions might be useful indicators for pre-dicting mortality. This information could be useful for fisheries management becausethere were no differences in the external morphological measurements between thesurvival groups in the present study, which suggests that, morphologically, therewere no visible differences between individuals that survived or died early. Theincorporation into fisheries science and management of rapid bioassay approaches,and use of field instruments which can provide immediate osmoregulatory and stressmetabolite information from plasma samples, have recently been advocated (Cookeet al., 2008; Hasler et al., 2009). Prespawning mortality can be substantial withinO. nerka populations in some years (Gilhousen, 1990), and en route migration mor-tality has been at 50–90% recently in some populations of Fraser River O. nerka(Cooke et al., 2004). Advanced warning of the extent or proportion of a populationthat may suffer prespawning or en route mortality could help managers in determin-ing whether to open or close in-river fisheries or in deciding how many fish to allowonto artificial spawning grounds.

The authors are grateful to E. Eliason, A. Lotto, K. Hruska, E. Martins, L. Pon, D. Roscoe,M. Hague, J. Lotto and Q. Renault for help with field and blood collections; also to J. Hills,V. Ives, M. Nomura and J. Carter for running the blood plasma assays; to T. Kozak forstatistical consultation; T. Clark for useful feedback on the manuscript; K. Charlie and theChehalis First Nation Band fishing crew; B. Smith from Fisheries and Oceans Cultus Lake


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Laboratory; M. Lapointe and S. Latham from the Pacific Salmon Commission and C. Wallace,J. Candy and the staff of the Molecular Genetics Laboratory at Fisheries and Oceans PacificBiological Station for the DNA analyses. This project was funded by a Natural Sciencesand Engineering Research Council of Canada (NSERC) strategic grant to S.G.H., K.M.M.and others, and from the Fisheries and Oceans Canada’s Environmental Watch programme.K.M.J. was supported by a NSERC postgraduate scholarship and a Pacific Century GraduateScholarship.

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