AN ABSTRACT OF THE THESIS OF
Cameron Saunders Sharpe for the degree of Master of Science
in Fisheries and Wildlife presented on September 10,
1992.
Title: Genetic and Environmental Variation in Stress
Physiology Among Steelhead Trout (Oncorhynchus mykiss)
Abstract approved:_
Redacted for Privacy
Carl B. Schreck
Juvenile steelhead trout (Oncorhynchus mykiss) of
different genetic origins exhibited different glucocorticoid
and lactate responses to stress.
Steelhead trout milt and
unfertilized eggs were obtained from three geographically
dispersed hatcheries in Oregon: Cole Rivers Hatchery
Applegate stock (CR), Marion Forks Hatchery Santiam stock
(MF), and Wallowa Hatchery Little Sheep Creek stock (LSC).
Gametes were transported to a rearing facility near
Corvallis, Oregon for rearing to experimental size in a
common environment.
Each stock was represented by the
progeny of 15 males mated to 15 females.
Survival from eyed
eggs to onset of feeding was 84%, 85% and 86% for CR, MF,
and LSC, respectively.
Electrophoretic analysis showed that
the three populations reared in a common environment closely
resemble their peers reared at their respective hatcheries.
Fish from the three hatchery stocks were subjected to
chronic confinement at their respective hatcheries and
sampled over 24 hr.
Three groups of fish from the same
hatcheries but reared from conception in a common
environment were similarly subjected to confinement and
sampled over 72 hr.
The cortisol response to stress varied
widely among groups of fish reared at their hatcheries and
in the common environment.
The lactate response to
confinement among fish reared at their hatcheries varied
Among stocks reared in a common
widely among hatcheries.
environment, variation in the lactate response to stress was
less evident than (but consistent with) trends established
for the cortisol response.
The patterns of variation among
stocks reared at their hatcheries and in the common
environment suggested that there is a genetic as well as an
environmental basis to the physical response to stress and
that environmentally induced variation can mask genetic
variation among stocks.
Differences in plasma cortisol
titers following stress may be related to differences among
stocks in secretory ability of interrenal tissue as shown by
incubation of anterior kidneys in defined media with
porcine-ACTH.
A protocol was developed to characterize catabolism of
cortisol by liver tissue of rainbow trout in vitro.
Minced,
washed liver tissue was incubated in defined media
containing cortisol and 1,2, 6,7-3H-cortisol.
Media and
tissue were sampled over time and putative products of
cortisol catabolism were characterized by HPLC.
A steroid
tentatively identified as cortisone and two other
unidentified steroids accumulated in media but most of the
derivatives of labeled cortisol remained in aqueous solution
after attempted extraction with dichloromethane.
This
suggests that conjugated derivatives of cortisol were
generated in vitro.
Genetic and Environmental Variation in Stress Physiology
Among
Steelhead Trout (Oncorhynchus mykiss)
by
Cameron Saunders Sharpe
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed 10 September 1992
Commencement June 1993
APPROVED:
Redacted for Privacy
Professor of Fisheries in charge of major
Redacted for Privacy
Head of Department of Fisheries and Wildlife
Redacted for Privacy
Dean of Grad
School
(I
Date thesis is presented: 10 September 1992
Typed by Cameron Saunders Sharpe
Acknowledgments
I wish to thank my major professor Dr. Carl B. Schreck
for his guidance throughout this study.
I also wish to thank all of my colleagues in the Oregon
Cooperative Fisheries Research Unit who offered support and
encouragement especially Mr. Kenneth P. Currens, Dr. Martin
Fitzpatrick, Dr. Alec Maule, Mr. Grant Feist, Mr.
Caleb
Slater, Dr. Hiram Li and, Mr. Steven L Stone.
Without the support and, above all, patience,
wife, Terri,
I could not have done any of this.
of my
TABLE OF CONTENTS
I. GENERAL INTRODUCTION
II. Stress Induced Changes in Circulating Levels of
Cortisol and Lactate and In Vitro Cortisol
Dynamics in 3 Stocks of Steelhead Trout
(Oncorhynchus mykiss) Reared at Their Hatcheries
and in a Common Environment
INTRODUCTION
1
7
8
MATERIALS AND METHODS
11
RESULTS AND DISCUSSION
22
CONCLUSIONS
53
BIBLIOGRAPHY
56
In vitro Cortisol Clearance and
Appendix A-2.
Metabolism in Steelhead Trout (Oncorhynchus
mykiss)
61
INTRODUCTION
61
METHODS
63
RESULTS AND DISCUSSION
66
CONCLUSIONS
70
LIST OF FIGURES
Cortisol concentration following chronic
confinement of steelhead trout at Cole Rivers
(CR), Marion Forks (MF), and Little Sheep Creek
(LSC) hatcheries, respectively.
Figure 1.
23
Cortisol concentration following chronic
Figure 2.
confinement of Cole Rivers (CR), Marion Forks
(MF), and Little Sheep Creek (LSC) steelhead trout
reared in the common environment at Smith Farm.
26
Lactate concentration following chronic
Figure 3.
confinement of steelhead trout at Cole Rivers
(CR), Marion Forks (MF), and Little Sheep Creek
(LSC) hatcheries, respectively.
29
Lactate response to chronic confinement of
Figure 4.
Cole Rivers (CR), Marion Forks (MF), and Little
Sheep Creek (LSC) steelhead trout reared in the
common environment at Smith Farm.
31
.
Plasma cortisol titer (mean + SE) in fish
continually confined for 72 hr and then sampled
before and 1 hr after imposition of a 30 second
acute handling stress. N=6 for each bar.
34
Plasma lactate titer (mean + SE) in fish
continually confined for 72 hr and then sampled
before and 1 hr after imposition of a 30 second
acute handling stress
36
In vitro secretion of cortisol by head
kidneys from Cole Rivers (CR) and Little Sheep
Creek (LSC) steelhead trout reared at their
respective hatcheries.
38
Figure 5.
.
.
Figure 6.
Figure 7.
In vitro secretion of cortisol by head
Figure 8.
kidneys from Cole Rivers (CR), Marion Forks (MF)
and Little Sheep Creek (LSC) steelhead trout
reared in the common environment.
41
Relationship between resting in vivo
cortisol levels and in vitro secretion of cortisol
by the head kidney over 2.5 hr in tissue culture
media without pACTH
43
Relationship between head kidney dry weight
Figure 10.
and in vitro secretion of cortisol by the head
kidney over 2.5 hr in tissue culture media without
pACTH
45
Figure 9.
Relationship between head kidney dry weight
Figure 11.
and fish size in fish used to test for a
relationship between size of the head kidney and
resting cortisol secretion in vitro
47
Dendrogram and Nei's unbiased genetic
Figure 12.
distance and identity matrix for Cole Rivers (CR),
Marion Forks (MF), and Little Sheep Creek (LSC)
steelhead trout reared at their hatcheries and in
a common environment.
49
Abundance (uCi) of radio-labeled cortisol
Figure 13.
and cortisol derivatives over time (1) extracted
with dichloromethane from incubation media
(MEDIA), (2) extracted from liver tissue (LIVER),
(3) remaining in aqueous phase after organic
extraction (AQUEOUS), and (4) total radioactivity
accounted for (TOTAL) over 24 hr incubation.
67
Genetic and Environmental Variation in Stress Physiology
Among
Steelhead Trout (Oncorhynchus mykiss)
I.
GENERAL INTRODUCTION
In salmonids, physiological indices of the response to
perturbations in their environment are extremely variable
among species, different genetic groups, and individuals
(Barton 1986).
The variation is dependent upon the nature
of the perturbation and upon intrinsic characteristics of
the animals themselves including genotype, ontogenetic
stage, and developmental history (Schreck 1981).
The
primary objective of this study is to determine if steelhead
trout of different genetic origins exhibit substantial
physiological differences in terms of their responses to
stress.
This study examines physiological variation within
steelhead trout (Oncorhynchus mykiss) using fish of diverse
geographic origin incubated, hatched and reared in a common
environment.
Selye (1973) defined stress as "the nonspecific
response of the body to any demand placed upon it" and
argued that this stress response was equivalent to the
General Adaptation Syndrome which would be elicited from any
organism subjected to stress.
He described the syndrome as
being comprised of three stages:
(1) the alarm phase when a
2
stimuli is recognized as a threat to homeostasis,
(2) the
resistance phase when an organism attempts to reestablish
and maintain homeostasis and, failing that,
(3) the
exhaustion phase when stress is severe or prolonged.
For
fish, this model has since been refined to limit the
response to stimuli that elicit fright, discomfort, or pain
(Schreck and. Lorz 1978; Schreck 1981).
this study,
For the purposes of
I will follow the advice of Pickering (1981)
with "stress" taken to mean the stimulus and the stress
response will be the reaction of the animals to the
stimulus; I will employ two widely applied physiological
measures of the stress response
circulating levels of
cortisol and lactate.
Cortisol elaboration in teleosts results from
activation of the hypothalamic-pituitary-interrenal axis and
is a primary indicator of the stress response.
Exposure to
a stress stimulates secretion of corticotropin releasing
factors (CRFs) by the hypothalamus (Fryer and Lederis 1986).
CRFs stimulate secretion of adrenocorticotropic hormone
(ACTH) by the pituitary (Donaldson 1981) which in turn
incites production of cortisol by interrenal tissue in the
head kidney.
Lactic acid is the main product of anaerobic glycolysis
(Heisler 1979) and, in teleosts, occurs primarily in white
muscle tissue (Holeton 1979), secondarily in liver (Mazeaud
and Mazeaud 1981).
Lactate is considered a secondary or
3
metabolic indicator of stress and lactate level in plasma is
associated with the exertion characteristic of physically
disturbed animals.
There is a growing body of evidence for a genetic basis
for the physiological response to stress in fish.
et al.
Fevolden
(1991) provided evidence for a genetic basis to the
stress response in rainbow trout and conclusively
established high and low cortisol response lines in Atlantic
salmon (Salmo salar).
Barton et al.
(1986) noted
differences among chinook salmon stocks in the magnitude of
the cumulative response to serial stressors, although the
authors noted that the differences could be attributed to
either genetic variation or rearing history.
Woodward and
Strange (1987) documented differences in the physiological
response to stress between hatchery and wild populations of
rainbow trout, but again, much of the variation can probably
be attributed to different rearing environments.
Physiological differences in terms of genetically
determined tolerance limits to stress encountered in a
fish's physical environment ("natural" stress) have also
been examined.
Hirshfield et al.
(1980) compared
physiological tolerances to temperature extremes and low
oxygen concentrations in isolated populations of desert
pupfish and found differences that they interpreted in terms
of local adaptation.
Similarly, genetic variation among
4
brook trout populations in tolerance to low pH environments
has been demonstrated (Swarts et al. 1978).
The genetic basis for variation in glucocorticoid
dynamics following stress has been established for avian
taxa (Carsia et al. 1988, Japanese quail; Gross and Colmano
1971, Gross and Siegal 1981, domestic chicken).
Substantial variation among species within the genus
Oncorhynchus has been noted for the magnitude of the primary
and secondary stress responses as measured by circulating
levels of cortisol and glucose, respectively (Barton 1986).
This, in combination with the observations of Barton et al.
(1986) that variation exists among chinook salmon stocks in
terms of the response to multiple consecutive stressors,
suggests that it is fruitful to look for variation of
genetic origin in steelhead trout.
ACTH stimulates salmonid interrenal tissue to produce
cortisol (Donaldson 1981; Patin° and Schreck 1988), the
interrenal hormone frequently used to measure the severity
of the primary stress response in fish.
If different
populations exhibit a different hormonal response to stress,
this might be resolved by measuring in parallel the in vitro
capacity of the fish to produce cortisol in response to
ACTH.
That is, since cortisol is a major stress response
hormone, differences in the capacity to produce cortisol may
reflect differences in the response to stress.
5
Since the realized circulating cortisol level is also a
function of clearance, this study includes the development
of an assay to characterize clearance and possible
metabolism of cortisol by liver tissue in vitro.
In
mammals, the liver is a major site of cortisol conjugation
for excretion in urine and metabolism to other steroids
(Henderson and Kime 1987).
In teleosts, cortisol is
catabolized into other steroids, principally cortisone (Kime
1978; Patin() et al. 1985; Donaldson and Fagerlund 1968) with
evidence in vivo of conjugation to tetrahydro cortisol
derivatives (Columbo et al. 1971; Truscott 1979).
Kime
(1978), however, was unable to detect the formation of
conjugated derivatives of cortisol in vitro in northern pike
(Esox lucius), white perch (Perca flavescens) or the rainbow
trout.
Thus, there is some question of the ability to
demonstrate in vitro some aspects of liver function.
Physiological characters, as quantitative traits, can
be expected to be environmentally labile (Falconer 1981).
The overall strategy of my research program involves
identifying aspects of the physiological response to stress
that vary substantially either among hatchery reared
populations or among the same groups reared in a common
environment.
If variation among populations reared at their
respective hatcheries is also evident among fish from the
same populations but reared in a common environment, a
6
likely interpretation is that some portion of the variation
is genetic in origin.
The thesis is organized into chapters.
the introduction to the thesis topic.
Chapter I is
Chapter II covers
stress physiology in general and presents evidence for a
genetic basis to variation in the stress response among
stocks.
The thesis chapters are followed by an appendix
describing the development of an in vitro assay system to
characterize catabolism of cortisol by liver tissue in
rainbow trout.
7
II. Stress Induced Changes in Circulating Levels of Cortisol
and Lactate and In Vitro Cortisol Dynamics in 3 Stocks of
Steelhead Trout (Oncorhynchus mykiss) Reared at Their
Hatcheries and in a Common Environment
8
INTRODUCTION
Genetic population structure in anadromous salmonids is
usually defined according to patterns of variation in
morphological and biochemical traits.
Relatively little
work has been done describing variation in traits correlated
with fitness.
In salmonids, physiological indices of the response to
perturbations in their environment are extremely variable
among species, different genetic groups,
(Barton 1986).
and individuals
The variation is dependent upon the nature
of the perturbation and upon intrinsic characteristics of
the animals themselves including genotype, ontogenetic
stage, and developmental history (Schreck 1981).
Herein I
examine variation within steelhead trout (Oncorhynchus
mykiss) from a physiological perspective using fish of
diverse geographic origin incubated, hatched and reared in a
common environment.
The primary objective of this chapter
is to determine if variation exists among stocks in terms of
stress physiology, a set of physiological parameters clearly
of substantial consequence to a fish's well being.
Cortisol production in teleosts is an indicator of the
primary stress response and results from activation of the
hypothalamic-pituitary-interrenal axis.
Imposition of a
stress stimulates secretion of corticotropin releasing
factors (CRF) by the hypothalamus (Fryer and Lederis 1986).
9
ORE stimulates secretion of adrenocorticotropic hormone
(ACTH) by the pituitary which in turn incites production of
cortisol by interrenal tissue in the head kidney.
Lactic acid is the main product of anaerobic glycolysis
(Heisler 1979) and, in teleosts, occurs primarily in white
muscle tissue (Holeton 1979), secondarily in liver (Mazeaud
and Mazeaud 1981).
Lactate is considered a secondary or
metabolic indicator of stress and lactate level in plasma is
associated with the exertion characteristic of physically
disturbed animals.
The particular genotype at one locus, LDH-B.2* (Lactate
Dehydrogenase, E.C. 1.1.1.27), has been associated with
variation in swimming performance in steelhead trout
(Tsuyuki 1977,).
In that work, some fish homozygous for the
176' allele (LDH-B.2* 76/76, formerly ldhH aBaB) exhibited
superior swimming stamina relative to fish homozygous for
the 1100' allele (LDH-B.2* 100/100, formerly ldhH
aAaA)
.
Presumably, LDH genotype affects clearance of the lactate
generated as a consequence of physical exertion and thus may
be a factor affecting the performance capacity of these
animals.
There is a growing body of evidence for a genetic basis
for the physiological response to stress in fish.
et al.
Fevolden
(1991) provided evidence for a genetic basis to the
stress response in rainbow trout and conclusively
established high and low cortisol response lines in Atlantic
10
salmon (Salmo salar).
Barton et al.
(1986) noted
differences among chinook salmon stocks in the magnitude of
the cumulative response to serial stressors, although the
authors noted that the differences could be attributed to
either genetic variation or rearing history.
Woodward and
Strange (1987) documented differences in the physiological
response to stress between hatchery and wild populations of
rainbow trout, but again, much of the variation can probably
be attributed to different rearing environments.
11
MATERIALS AND METHODS
Experimental animals and rearing conditions: Steelhead
trout milt and unfertilized eggs were obtained from three
Oregon Department of Fish and Wildlife hatcheries;
Marion
Forks Hatchery Minto trap Santiam stock (MF) on the North
Santiam River, the Cole Rivers Hatchery Applegate stock (CR)
on the Rogue River, and the Wallowa Hatchery Little Sheep
Creek trap (LSC).
Electrophoretic comparisons of allozymes
(Schreck et al. 1986; Hatch 1990) suggest that fish from
these three sources can be considered representative of
different genetic groups.
Throughout rearing, every effort
was expended to maintain homogeneity among environments.
Gametes from MF, CR, and LSC were obtained on May 3, 4, and
5 1988, respectively.
This should have eliminated run
timing as a variable and permitted synchronous development
of all fish apart from maternal effects and genetically
determined differences in growth.
Eggs from each female
were stored separately in plastic bags and transported at
about 2 C to Smith Farm, our rearing facility near
Corvallis, Oregon.
Milt was collected in test tubes, stored
in oxygen filled plastic bags and transported at about 2 C.
All gametes were held for 6 hr prior to fertilization to
minimize the potential for differential transport effects
among stocks.
The eggs from one female fertilized with the milt from
one male yielded one family.
Each group of experimental
12
animals was represented by 15 families.
Each family was
held in separate incubation cells distributed throughout a
Heath Tray incubator with a flow rate of 7 liters per minute
at 11 C.
The Heath trays were flushed daily with a
malachite green fungicide solution.
When most of the eggs
became eyed, the eggs were shocked and unfertilized or dead
eggs were removed.
After hatch, each family was split into
2 incubation cells to avoid crowding.
Prior to onset of
feeding equal numbers of fish from each family were pooled
within each stock.
Each pool was split into four equal
groups and each group was transferred into a 14 1 circular
flow through tank for rearing.
Fish in the 14 1 tanks were
fed Oregon Moist Pellet (OMP) between 4 and 6 times daily.
After rearing to about 3 grams, fish from each stock were
distributed into 1 m diameter circular flow through tanks
(2
tanks/stock) for final rearing to a convenient experimental
size.
These fish were fed OMP daily.
The experiments performed at the hatcheries used fish
from the same brood year as the fish reared at Smith Farm.
They represent peers and sibs of the fish reared in the
common environment.
Electrophoresis:
In order to ascertain whether fish
conceived and reared at Smith Farm are genetically
representative of their peers reared at their respective
hatcheries, electrophoretic analysis, following the
methodology of Aebersold et. al (1987), was performed.
13
Eighty fish from each stock from each of the chronic
confinement experiments were frozen on dry ice for starch
gel electrophoresis.
Samples of liver and white muscle
tissue were electrophoresed to establish genotype at 18 loci
(Table 1)
for each fish.
BIOSYS-1 (Swofford and Selander
1989) was used for analysis.
A locus was considered
polymorphic when the frequency of the common allele was less
than 0.99 in one or more of the groups tested.
Allele
frequencies at loci polymorphic in any of the groups were
used to compute Nei's unbiased genetic distance and identity
measures for comparison (1) between groups reared in
different environments,
(2) between stocks reared at their
respective hatcheries and (3) between stocks reared in a
common environment.
Stress response treatment:
Evaluation of the response
to a chronic stress, confinement, followed the methodology
of Barton (1986).
The first experiment was conducted at the
hatcheries from March 7 to March 14 1989 using MF,
LSC steelhead trout (7.9+0.3g,
respectively).
CR and
42.7+11.2g, and 53.6+15.0g,
Duplicate groups of steelhead, 50 fish per
stock per replicate, were placed in perforated buckets
submerged in the raceways to a depth just sufficient to
cover the fish and the fish were then sampled after 0.5,
3,
6,
9, and 24 hr.
1,
Control (unconfined) fish were sampled
from the raceways just before and 24 hr after the rest of
the fish were confined.
Depth of each bucket was adjusted
14
Table 1.
International Union of Biochemistry (I.U.B.)
enzyme names, Enzyme Commission (E.C.) numbers, loci
examined in this study, tissues, and buffers.
muscle; L, liver.
Buffers: TBCL
Tissues: M,
a Tris-citrate gel buffer
at pH 8.5; TBE a Tris-borate-EDTA gel and tray buffer at pH
8.5; and ACE
pH 7.0.
an amine citrate-EDTA gel and tray buffer at
An asterisk indicates that the system was
polymorphic in one or more of the groups tested.
15
Table 1.
I.U.B. Enzyme Name
E.C. Number
Aconitate hydratase
Alcohol dehydrogenase
Aspartate aminotransferase
Creatine kinase
4.2.1.3
1.1.1.1
2.6.1.1
2.7.3.2
Glucose-6-phosphate
isomerase
5.3.1.9
Glycerol-3-phosphate
dehydrogenase
L-lactate denydrogenase
Malate dehydrogenase (NADP+)
Dipeptidase
Tripeptide aminopeptidase
Phosphoglucomutase
Superoxide dismutase
Locus
sACOH*
ADH*
sAAT-2.1,2*
CK-Al*
CK-A2*
GPI-A*
GPI-B1*
GPI-B2*
G3PDH-1.1*
G3PDH-1.2*
1.1.1.27 LDH-B2*
1.1.1.40 sMDHP-1,2*
mMDHP-1,2*
3.4.13.11 PEP-A*
3.4.11.4 PEP-B*
PGM-1.1*
5.4.2.2
PGM-1.2*
1.15.1.1 sSOD*
1.1.1.8
Tissue
L
L
M
M
M
M
M
M
M
M
L
M
M
M
M
M
M
L
Buffer
TBCL
ACE
ACE
TBCL
TBCL
TBCL
TBCL
TBCL
ACE
ACE
TBCL
ACE
ACE
TBE
TBE
ACE
ACE
TBCL
16
after 3,
6, and 9 hr to maintain an approximately constant
density.
Each sample consisted of six fish in duplicate
except for control samples which consisted of three fish in
duplicate.
Sampling proceeded as follows: for each sample, fish
were quickly netted and killed in a lethal concentration of
buffered 3-aminobenzoic acid ethyl ester methanesulfonate
salt (MS222: 200 mg/1).
Weight was noted and blood was
collected in ammonium-heparinized capillary tubes after
severing the caudal peduncle.
After centrifugation the
Plasma cortisol levels were obtained by
plasma was frozen.
radioimmunoassay (RIA) using the assay protocol developed by
Foster and Dunn (1974) as modified by Redding et al.
(1984).
Plasma lactate levels were obtained using the fluorimetric
method (Passonneau 1974).
The experiment was repeated on April 17 through April
20, 1989 using fish from the three stocks but reared in the
common environment.
At Smith Farm, the confinement buckets
were hung in a single 2.13 m circular tank and the duration
of the experiment was extended with additional samples taken
at 48 and 72 hr.
CR, MF, and LSC fish were 15.3+0.6g,
15.5+0.6, and 16.0+0.6g, respectively, at the time of the
experiment.
Sample size for MF and LSC trout was five fish
in duplicate for stressed fish and prestress controls (t =
0
hr) and three fish in duplicate for unstressed controls (t =
24, 48, and 72 hr) .
17
Sample design for CR was as above except that at 72 hr
only 4 fish in duplicate were sampled.
By 72 hr, both cortisol and lactate titers were
expected to decline from peak levels.
In order to determine
if this decrease was associated with acclimation or
exhaustion, fish remaining in the buckets after 72 hr of
continuous confinement were subjected to an acute stress and
sampled 1 hour thereafter.
The acute stress was imposed by
suspending the buckets in air for 30 seconds and then
returning them to the water.
Sample size for each stock was
3 fish in duplicate.
In vitro incubations:
These experiments involved the
generation of dose response curves by static incubation of
interrenal tissue from juvenile steelhead trout (parr) in
tissue culture media (TCM) containing various doses of
porcine ACTH (pACTH; Sigma).
of Patin° et al.
The methodology followed that
(1986) with some modification.
For each
experiment, the anterior kidney was removed from each fish
to ice cold TCM, minced, and washed twice (2 hr per wash) in
TCM.
Tissues were then incubated for 2.5 hr in TCM and an
aliquot was removed to establish resting cortisol secretion
for each head kidney.
The tissues were then transferred to
TCM containing pACTH and incubation was continued for
another 2.5 hr.
Preliminary work in this laboratory showed
that resting cortisol secretion and sensitivity to pACTH
does not change in juvenile steelhead trout head kidneys
18
even after a 4 hr preincubation.
Sample sizes for each
replicate were 4 fish for each of 5 concentrations of pACTH
plus head kidneys of 4 fish incubated in TCM without pACTH
to establish resting cortisol secretion rate.
The stock
solution of pACTH was prepared by reconstituting a single
vial of the hormone in TCM and distributing aliquots into
microcentrifuge tubes individually frozen at -80 C.
Preliminary experiments using pACTH concentrations of
0.0125, 0.125, 1.25, 6.25, and 12.5 mIU pACTH /ml showed that
this series best characterized the dose response of juvenile
steelhead trout interrenal tissue to pACTH.
These
preliminary experiments were also used to ascertain whether
sex, fish size, or size of the head kidney might confound
results of in vitro incubations.
Each fish was weighed at
the time of dissection and sex established by inspection.
Wet and dry weights of the head kidney tissue was recorded
after all washes, incubations, and sampling.
In February 1989, responsivity of interrenal tissue of
fish from CR, MF, and LSC
was evaluated.
reared in a common environment
The experiment was repeated at Cole Rivers
and Irrigon hatcheries using their resident CR and LSC fish,
respectively, in March 1989.
Washes and incubations were
carried out on an orbital shaker at 100 RPM in CorningR
tissue culture plates at constant temperature (12 C) in a
refrigerator at our laboratory and a portable KoolatronR
cooler at the hatcheries.
Following incubation, an aliquot
19
of TOM was removed from each sample and frozen at -80 C in
Cortisol
our laboratory and on dry ice at the hatcheries.
titer for each sample was obtained by RIA using the
procedure described previously for plasma except that the
standards were reconstituted in TCM after Patin() et al.
(1986)
.
In order to ascertain whether a relationship existed
between in vitro resting cortisol secretion and circulating
levels of cortisol in unstressed fish, plasma samples were
taken just before removal of the head kidneys.
Only the
fish reared in the common environment were sampled for this
portion of the experiment.
MF, and 16 for LSC.
Sample sizes were 24 for CR and
Cortisol titer in plasma was
established by RIA as described.
Electrophoretic analysis:
Gametes were taken once from
each hatchery because of experimental constraints, space
limitations of our rearing facility and the logistic
difficulties of obtaining multiple samples from
geographically dispersed hatcheries.
A possible
consequence, however, is that the Smith Farm fish were not
representative of each hatchery's entire spawning run
In order to ascertain whether fish conceived and reared
at Smith Farm are genetically representative of their peers
reared at their respective hatcheries, electrophoretic
analysis, following the methodology of Aebersold et. al
(1987), was performed.
Eighty fish from each stock from
20
each of the chronic confinement experiments (Chapter 1) were
retained, frozen on dry ice, and transported back to the
laboratory for starch gel electrophoresis.
Samples of liver
and white muscle tissue were electrophoresed to establish
genotype at 18 loci (Table 1) for each fish.
BIOSYS-1
(Swofford and Selander 1989) was used for analysis.
A locus
was considered polymorphic when the frequency of the common
allele was less than 0.99 in one or more of the groups
tested.
Allele frequencies at loci polymorphic in any of
the groups were used to compute Nei's unbiased genetic
distance and identity measures for comparison (1) between
groups reared in different environments,
(2) between stocks
reared at their respective hatcheries and (3) between stocks
reared in a common environment.
Circulating levels of lactic acid during chronic
confinement were established for each of the fish as were
the electrophoretic phenotypes for LDH-B.2* for each fish.
In order to ascertain the effects of LDH genotype on lactate
dynamics in plasma, lactate data from the chronic
confinement experiments were sorted according to LDH-B.2*
genotype and the resulting response curves tested for
differences, attributable to genotype, in the lactate
response to chronic confinement.
Furthermore, the size at age was known for each fish
reared at Smith Farm at the time of sampling for
electrophoresis.
In order to ascertain whether genotype at
21
any of the polymorphic loci might be related to growth, size
data from fish sampled for electrophoresis were sorted
according to genotype at polymorphic loci and compared for
differences in size at age potentially attributable to
genotype.
Statistics:
For both the chronic confinement and pACTH
dose response experiments, some of the cortisol sample
distributions were not normal nor were sample variances
equal.
The lactate sample distributions from the chronic
confinement experiment were similarly problematic.
Log
transformation of the cortisol and lactate data was used to
normalize the data and decrease heterogeneity among
variances.
Cortisol and lactate response curves were tested
for significant differences between replicates.
Replicates
in the chronic confinement experiments did not differ
significantly (P>0.05) and subsequent tests were performed
on pooled data. Cortisol and lactate response curves were
tested for significant differences (P<0.05) between stocks
by ANOVA followed by Duncan's multiple range test.
Furthermore, for the pACTH dose response experiments,
t-tests were used to ascertain whether differences
attributable to sex existed in levels of cortisol secreted
by unstimulated head kidneys.
Regression analysis was used
to test for variation in resting cortisol secretion
attributable to fish size and size of the head kidney.
22
RESULTS AND DISCUSSION
Response to chronic confinement:
Steelhead trout
exhibit significant variation among stocks in terms of how
the fish from each stock respond when chronically confined.
The variation can be explained by genetic differences among
stocks and differences in rearing history of fish at
different hatcheries.
MF steelhead attained and maintained higher plasma
cortisol levels than CR or LSC trout when the fish were
continuously stressed at their respective hatcheries
1).
(Figure
The patterns of cortisol titer over the course of the
experiments were similar for CR and MF trout
increase to a maximum at 9 hr.
a gradual
Cortisol levels in LSC fish
differed from those of CR and MF fish in that the cortisol
titer peaked at 1 hr, decreased significantly at 3 hr, and
then remained relatively constant in subsequent samples.
In
all cases, cortisol titer was low before imposition of
stress and in unstressed controls sampled at 24 hr.
Variation in rearing environment clearly could have
played a role in generating the variation among stocks noted
when fish were reared at their respective hatcheries.
McLeay (1975) reported activation of the pituitaryinterrenal axis by cold water acclimation in juvenile coho
salmon (0. kisutch).
At the hatcheries, MF fish were
acclimated to 5 C water while both LSC and CR fish were
acclimated to 11 C water.
Furthermore, at the time of the
23
Figure 1.
Cortisol concentration following chronic
confinement of steelhead trout at Cole Rivers (CR), Marion
Forks (MF), and Little Sheep Creek (LSC) hatcheries,
respectively.
Values on response curves are means + one SE.
N=12 for points on response curve.
at 24 H.
N=6 for control points
An "a" indicates that the mean cortisol titer of
that sample is significantly elevated (Duncan; P < 0.05)
over both other samples taken at that time.
A "b" indicates
that a sample is significantly elevated over the lowest
cortisol titer.
TIME (hr)
25
experiment, the Marion Forks Hatchery was experiencing
difficulties with river otters entering the raceways and
consuming fish.
Sometime between the 9 and 24 hr samples at
least one otter entered the raceway in which the confinement
buckets were suspended.
Some of the variation among stocks reared at their
hatcheries may be attributable to differences in smolt
development among stocks whereby animals at different states
of smolting had higher resting levels of cortisol and
responded differently to stress.
LSC fish were larger and
were released as yearlings in late April, 1989, while CR and
MF fish were released as two-year-olds in mid-April, 1990.
Resting cortisol level (Figure 1; t=0h), an index of
smoltification (Barton et al. 1985, Patin° et al. 1986), was
greater in LSC fish than CR and MF fish and circulating
cortisol levels in stressed LSC fish was greater at t=0.5h
and t=1.0h than in CR fish.
Differences were also noted among stocks reared in the
common environment and subjected to a chronic stress.
CR
trout attained higher cortisol titers and peaked later than
either MF or LSC fish (Figure 2).
Cortisol titers for MF
and LSC trout increased to a peak at 3 hr followed by a very
gradual decrease over the course of 3 days.
The pattern was
similar for CR fish except that the peak occurred at 9 hr.
None of the treatment groups ever reestablished the
consistently low cortisol levels of control fish.
26
Figure 2.
Cortisol concentration following chronic
confinement of Cole Rivers (CR), Marion Forks (MF), and
Little Sheep Creek (LSC) steelhead trout reared in the
common environment at Smith Farm.
are means + one SE.
Values on response curves
N=10 for points on response curves.
N=6 for control points at 24, 48 and 72 H.
An "a" indicates
that the mean cortisol titer of that sample is significantly
elevated (Duncan; P < 0.05) over both other samples taken at
that time.
A "b" indicates that a sample is significantly
elevated over the lowest cortisol titer.
350
300
250
200
150
100
48
TIME (hr)
72
28
Plasma lactate response curves varied significantly
among stocks of fish reared at their hatcheries (Figure 3).
In the chronic confinement experiment at the hatcheries,
plasma lactate rose rapidly, peaked in each case at 1 hr,
and returned to near resting levels after 6 hr of continuous
confinement.
The peak response and duration of that
response was significantly greater in LSC fish than in
either CR and MF fish.
The peak response in CR fish was
significantly greater than that of the MF fish.
Less variability in the lactate concentration in
response to chronic confinement was noted among stocks when
the fish were reared in a common environment than at their
respective hatcheries.
Again, plasma lactate peaked at 1 hr
and decreased to near resting levels by 6 hr (Figure 4).
Lactate levels in CR fish were greater than levels in LSC
fish at 24 and 48 hr and greater than levels in MF fish at
48 hr.
In th2. experiments with fish reared and chronically
stressed at their hatcheries, no mortalities were noted in
any of the treatment groups.
However, with fish reared and
chronically confined in the common environment, nine CR fish
died, seven between 0 and 24 hr and two more between 24 and
48 hr.
Two MF fish died between 24 and 48 hr and none of
the LSC fish died.
These differences are statistically
significant (X2=12.6; df=2) .
29
Figure 3.
Lactate concentration following chronic
confinement of steelhead trout at Cole Rivers (CR), Marion
Forks (MF), and Little Sheep Creek (LSC) hatcheries,
respectively.
Values on response curves are means + one SE.
N=12 for points on response curve.
at 24 H.
N=6 for control points
An "a" indicates that the mean lactate titer of
that sample is significantly elevated (Duncan; P < 0.05)
over both other samples taken at that time.
A "b" indicates
that a sample is significantly elevated over the lowest
lactate titer.
0
3
6
TIME (hr)
12
24
31
Figure 4.
Lactate response to chronic confinement of Cole
Rivers (CR), Marion Forks (MF), and Little Sheep Creek (LSC)
steelhead trout reared in the common environment at Smith
Farm.
Values on response curves are means + one SE.
for points on response curves.
24, 48 and 72 H.
N=10
N=6 for control points at
An "a" indicates that the mean lactate
titer of that sample is significantly elevated (Duncan; P <
0.05) over both other samples taken at that time.
A "b"
indicates that a sample is significantly elevated over the
lowest lactate titer.
0
3
6
9
TIME (hr)
24
48
72
33
After 72 hr of continuous confinement, fish given an
acute stress were still able to elaborate more cortisol
(Figure 5) suggesting that the descending arm of the
cortisol response curve reflects partial compensation to
confinement stress.
There were no statistically significant
differences in lactate levels before and after the acute
stress (Figure 6).
In general, the characteristics of the response curves
for cortisol and lactate (initial and peak values, duration)
are in agreement with values published in the literature
(see Barton et al. 1980; Barton 1986; Mesa and Schreck 1989,
for example).
Dose responses to pACTH:
In the dose response
experiments performed at the hatcheries, only CR and LSC
fish were used to characterize the in vitro response to
pACTH.
MF fish were so much smaller than CR or LSC fish
(7.9g+0.3, 42.7g+11.2, and 53.6g+15.0, respectively)
that no
meaningful comparisons could be made if MF fish had been
included in the experiment.
Very clear dose dependent
responses to pACTH were evident in the fish, however no
significant differences (ANOVA; P<0.95) were noted between
stocks (Figure 7).
The similarity of the dose responses between fish
reared at their respective hatcheries is not surprising in
the face of the results from the chronic stress experiment:
CR and LSC fish maintained similar plasma cortisol titers
34
Figure 5.
Plasma cortisol titer (mean + SE) in fish
continually confined for 72 hr and then sampled before and
hr after imposition of a 30 second acute handling stress.
N=6 for each bar.
1
35
IllCONFINEMENT
//.
CONFINEMENT + HANDLING
I
I
COLE
RIVERS
MARION
FORKS
I
LITTLE
SHEEP CR.
HATCHERY STRAIN
36
Figure 6.
Plasma lactate titer (mean + SE) in fish
continually confined for 72 hr and then sampled before and 1
hr after imposition of a 30 second acute handling stress.
N=6 for each bar.
37
I CONFINEMENT
12 CONFINEMENT+HANDLING
CR
MF
STOCK
LSC
38
Figure 7.
Concentration (mean + SEM) of cortisol secreted
in vitro by head kidneys from Cole Rivers (CR) and Little
Sheep Creek (LSO) steelhead trout reared at their respective
hatcheries.
N=4 for each bar.
39
200
COLE RIVERS
t150
.ti
%/
Z
-4
0
(J)
0
U
L. SHEEP CR.
100
50
0
0
0.0125
0.125
1.25
6.25
pACTH (mU/m1)
12.5
40
when chronically confined and possessed interrenal tissue
which elaborated similar quantities of cortisol in response
to pACTH.
The experiment was repeated using fish reared in a
common environment.
Dose dependent responses were evident
(Figure 8) but the magnitude of the responses differed
between replicates (ANOVA; P<.95) and the data could not be
pooled.
Thus, perhaps because of high variance and the
small sample size, no statistically significant differences
between stocks were noted.
No correlation was found between resting plasma
cortisol levels in vivo and cortisol levels secreted in
vitro by unstimulated head kidneys (Figure 9).
No relationship was found between resting secretion in
vitro and size of the head kidney (Figure 10) over the size
range of the fish tested even though head kidney size was
well correlated with fish size (Figure 11).
Electrophoresis:
Fish reared in a common environment
were genetically very similar to their hatchery-reared peers
and dissimilar to fish of different hatchery origins (Figure
12)
.
Nine of 18 enzyme systems were polymorphic in one or
more of the groups tested.
Resolution of enzyme activity
for these enzymes was adequate such that allele frequency
data could be used for analysis of genetic variation among
stocks.
41
Figure 8.
Concentration (mean + SEM) of cortisol secreted
in vitro by head kidneys from Cole Rivers (CR), Marion Forks
(MF) and Little Sheep Creek (LSC) steelhead trout reared in
the common environment.
Replicates are presented separately
because of significant differences (ANOVA; P<0.05) in the
magnitude of the responses between replicates.
250 -
LITTLE SHEEP
CREEK
MARION
COLE
RIVERS
225 -
FORKS
200 -
FIRST REPLICATE
175 -
El SECOND REPLICATE
150 125 -
100 -
75 -
50 25 0
am,
0
0.0125 0.125
1.25
6.25
12.5
0
0.0125 0.125
1.25
6.2;
pACTH (rnU/m1)
12.5
0
0.0125 0125
1.25
6.25
12.5
43
Figure 9.
Relationship between resting in vivo plasma
cortisol levels and in vitro secretion of cortisol by the
head kidney over 2.5 hr in tissue culture media without
pACTH.
44
y = 1.3665 + 6.0054e-2x RA2 = 0.022
0
5
10
15
IN VITRO CORTISOL SECRETION (ng/m1)
20
45
Figure 10.
Relationship between head kidney dry weight and
in vitro secretion of cortisol by the head kidney over 2.5
hr in tissue culture media without pACTH.
46
10
102 = 0.004
y = 1.7649 + 9.8932e-2x
8
6
s
OD
4
2
4011'
(DI.
0
0
r
z ).`
1
I
1
1
2
3
I
.
I
1
1
4
5
6
HEAD KIDNEY WEIGHT (mg)
7
47
Figure 11.
Relationship between head kidney dry weight and
fish size in fish used to test for a relationship between
size of the head kidney and resting cortisol secretion in
vitro.
48
y = 0.87183 + 0.16736x RA2 = 0.607
FISH WEIGHT (gm)
49
Figure 12.
Dendrogram and Nei's unbiased genetic distance
and identity matrix for Cole Rivers (CR), Marion Forks (MF),
and Little Sheep Creek (LSC) steelhead trout reared at their
hatcheries and in a common environment.
In matrix, genetic
distances are below and genetic identities are above the
diagonal.
Matrix is based upon allele frequencies of all
polymorphic enzyme systems.
50
NEI'S GENETIC DISTANCE
1
.10
1
.08
1
1
.06
.00
.02
.04
CR AT SF
CR AT CR
MF AT SF
MF AT MF
LSC AT SF
LSC AT LSC
POPULATION
1
1 CR AT SF
2 CR AT CR
3 MF AT SF
4 MF AT MF
5 LSC AT SF
6 LSC AT LSC
0.001
0.056
0.049
0.053
0.053
2
3
4
5
6
0.999
0.946
0.955
0.952
0.961
1.000
0.949
0.953
0.962
0.954
0.949
0.955
0.971
0. %3
0.995
0.046
0.040
0.048
0.046
0.000
0.039
0.030
0.047
0.038
0.005
51
Phenotype ratios for polymorphic systems did not
deviate from Hardy-Weinberg expectations in any of the
groups reared at their hatcheries.
In addition, all five of
the polymorphic systems in CR fish reared at Smith Farm were
in Hardy-Weinberg equilibrium.
However, of the other
animals reared in a common environment, an excess of
heterozygotes (P=0.013) was noted at one locus (sSOD*)
fish.
Also, a heterozygote deficiency at LDH-B2*
in MF
(P=0.040)
and a heterozygote excess at sACOH* (P=0.004) was noted in
LSC fish.
The deviation from Hardy-Weinberg expectations at LDHB2* in LSC fish may represent a statistical type 1 error
(Stahl 1987) but the very significant deviations at sSOD*
and sACOH* may reflect non-random mating as a result of the
small number of parents used to propagate the study animals.
Alternatively, the deviations from expected ratios may
reflect selection for particular genotypes at the loci in
question or inadvertent mixing among groups.
Mixing is an
unlikely explanation given other electrophoretic evidence.
For example, sMDHP* is quite variable in CR fish but fixed
for the most common allele in MF and LSC fish.
Similarly,
G3PDH-1,1* is variable in MF fish but fixed in CR and LSC
fish.
Had mixing occurred, allele frequencies would have
been more homogeneous among groups.
Another explanation for the heterozygote deficiency at
LDH-4* in LSC fish, may be that inbreeding could have
52
occurred if the animals ripe on the one day of sampling had
a tendency to be more closely related to each other than to
other animals in the cohort (Allendorf and Ryman 1987).
Allendorf and Ryman also suggest that the incidence of
heterozygote deficiency might follow from a tendency toward
differences in allele frequencies between sexes.
No relationships between genotype and size at age and,
more specifically, LDH-B2* genotype and lactate dynamics
were noted.
The interaction of LDH-B2* and lactate dynamics
deserves more careful study, however, given the findings of
Tsuyaki and Williscroft (1977).
It is possible, although
still speculative, that genotype at that locus is more
closely involved in regulation of lactate in tissue
compartments other than the blood.
For example, LDH-B2* is
expressed with other LDH* loci in muscle and is the only
LDH* expressed in liver.
53
CONCLUSIONS
The variation among stocks reared in a common
environment suggested a substantial genetic component
controlling the magnitude of the glucocorticoid response to
stress.
Of the stocks reared at their respective
hatcheries, LSC fish exhibited the highest initial cortisol
titers but after prolonged confinement MF fish attained
higher cortisol titers than either CR or LSC fish.
After rearing in a common environment, CR fish were
demonstrably more responsive and sensitive to chronic
confinement: cortisol and, to a lesser extent lactate,
titers were higher in CR fish and mortality was greater.
A
plausible explanation for the observation that the relative
responsivity and sensitivity to chronic confinement differs
among stocks depending upon how the animals are reared is
that the environmental component controlling the magnitude
of the stress response in steelhead trout masked the effect
of the genetic component.
Physiological variation among stocks was noted and,
since the animals were reared in a common environment,
I
infer that some of the variation may then be attributable to
genetic differences.
The strength of that inference is
compromised by the distinction between common and identical
environments: for the fish reared at our experimental
hatchery, every effort was made to create homogeneous
54
environments among stocks but subtle differences in rearing
histories might have contributed to the observed variation
in stress physiology.
al.
In mammals, for example, Meaney et
(1987) noted that repeated handling of neonatal rats
caused changes in the adrenocortical axis that were
persistent throughout life.
Furthermore, the possibility that maternal effects
contributed to variation among groups was not explored.
An
investigation of that aspect of inheritance was not possible
within the logistic constraints of this work but deserves
study.
Management implications following from the observation
that animals differ in their response to stress are
threefold.
First, genetic variation may have exploitable
heritability and fish culturists could benefit from the
selective breeding of stress resistant strains.
Presumably,
selective breeding would be especially useful for
aquacultural practices generating intensively cultured fish
not intended for release as in net-pen rearing.
Second, aquaculturalists could benefit from the
potential for variation of environmental origin by
manipulating the hatchery environment.
Animals could be
conditioned to stress to reduce the negative impacts of
unavoidable handling, crowding, tagging, and transportation
of hatchery fish.
55
Finally, the stress response is a single, albeit
critical, aspect of physiology.
Given that it is likely
that other performance traits express similar patterns of
variation among stocks, existing, essentially neutralist,
methods for differentiation among stocks (allozyme and RFLP
analyses) could be complemented by characterization of
genetic population structure on the basis of patterns of
functional, physiologically relevant variation.
56
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copper and potential use of cortisol as an indicator of
Journal of the Fisheries Research Board of
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W.H. Freeman and Company, San Francisco, California.
859 pp.
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Specker, J.L. and C.B. Schreck. 1982. Changes in plasma
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Appendix
61
Appendix A-1.
In Vitro Cortisol Clearance and Metabolism in
Steelhead Trout (Oncorhynchus mykiss)
INTRODUCTION
In salmonids, the glucocorticoid response to stress is
extremely variable among species, subspecies, races, and
individuals.
Circulating level of cortisol has frequently
been used as a measure of the degree to which animals have
been disturbed by perturbations in their environment and has
been associated with variation in performance capacity of
the animals.
Evolutionary and environmental forces
certainly contribute to the variation but actual
physiological mechanisms driving variation in glucocorticoid
dynamics following stress are poorly understood.
The circulating level of cortisol in teleosts is a
function of activation of the hypothalamic-pituitaryinterrenal axis by stress, for example, but the dynamics of
the glucocorticoid response
its magnitude and duration
must also be shaped by steroid clearance mechanisms.
Potentially, variation among individuals in the
glucocorticoid response to stress could be attributed to
differences among individuals in steroid metabolic clearance
rates.
In mammals, the liver is a major site of cortisol
conjugation for excretion in urine and metabolism to other
steroids (Henderson and Kime 1987).
In teleosts, cortisol
62
is catabolized in vivo into other steroids, principally
cortisone (Kime 1978; Patina et al. 1985; Donaldson and
Fagerlund 1968) and to a host of hydrolyzable conjugates
(Columbo et al. 1971; Truscott 1979).
Kime (1978), however,
was unable to detect in vitro formation of conjugated
derivatives of cortisol in northern pike (Esox lucius),
white perch (Perca flavescens) or the rainbow trout
(Oncorhynchus mvkiss).
The purpose of this work is to examine cortisol
clearance in rainbow trout by developing an in vitro
protocol involving incubation of liver tissue in defined
media with cortisol and 1,2, 6, 7-3H-cortisol.
I expect that
experimental application of this protocol will include
investigations into how cortisol clearance is related to
cortisol dynamics following stress and to how variation
among individuals in metabolic clearance rate is involved in
driving differences among individuals in the glucocorticoid
response to stress.
63
METHODS
In vitro incubation:
In April 1990, a single juvenile
rainbow trout (490 gm) reared from gametes obtained from the
Cole Rivers Hatchery (Oregon) was killed in buffered MS222
(200 mg /l) and the liver sans gallbladder and an
approximately 5 gm piece of white muscle tissue was removed
to ice cold tissue culture media (TCM; Patin° et al. 1986).
The liver and muscle samples were finely minced and
transferred to 5 ml TCM and washed under blood gas (10% 02,
10% 002, 80% N2)
two hr.
at 12 C on an orbital shaker (100 rpm) for
The wash was repeated and then the liver and muscle
fragments were separated into four 1 gm portions.
Each
portion was placed into one of four incubation wells with 5
ml TCM containing cortisol (10-5M) and 1,2, 6, 7-3H-cortisol
(20,000 cpm/ml).
Tissue and 4 ml media were immediately
sampled from one of the wells as a control.
The remaining
three wells were incubated under conditions identical to
that described for the washes.
Tissue and media were
sampled from the second well after 3 hr, the third after 6
hr, and the fourth after 24 hr.
In addition to the t=0
media and tissue samples, other control samples included (1)
a media sample without cortisol or 1,2, 6, 7-3H-cortisol
added,
(2)
a media blank containing cortisol and 1,2,6,7-3H-
cortisol but no tissue incubated 24 hr.
Extractions:
At the time of each sample, tissue and a
4 ml aliquot of media were separately frozen at -80 C so
64
that all extractions could take place at one time.
The
tissue samples and four 1 ml aliquots of each media sample
were extracted separately with three 5 ml volumes of
dichloromethane.
To account for tritium not extractable by
dichloromethane, the tissues were solubilized in ProtosolR
(vendor) and counted in BudgetsolveR (vendor) scintillation
cocktail on a Beckman LS1800 scintillation counter.
Media
extracts of each sample were pooled, evaporated to dryness
under vacuum, reconstituted in 1 ml methanol and filtered
through a 0.45 micron filter.
Samples were again evaporated
to dryness under vacuum and reconstituted in 60 ul mobile
phase (62% dddH2O, 28% methanol, 5% acetonitrile, 5%
propanol) for separation on reverse phase HPLC employing 254
and 280 nm detectors following the methodology of Huang et
al.
(1983) as modified by Feist et al.
(1990).
A 50 ul
aliquot of each sample was injected onto the column and 1
min fractions were collected.
Each fraction was counted in
BudgetsolveR (vendor) scintillation cocktail on a Beckman
LS1800 scintillation counter.
A standard cocktail
containing aldosterone, cortisone, cortisol, 11ketotestosterone, 11-8-0H-androstenedione, 11-8-0Htestosterone, corticosterone, 11-deoxycortisol, 11ketoprogesterone, androstenedione, estradiol, testosterone,
17-0H-progesterone, 17a- 2013- dehydroxy- progesterone,
progesterone, and 208-OH-progesterone was injected unto the
column after every 2 samples to facilitate identification of
65
some of the putative products of in vitro cortisol
metabolism.
Extraction efficiencies:
Strong evidence exists for
formation of hydrolyzable conjugates of cortisol in vivo
(Truscott 1979) in rainbow trout.
Thus, glucocorticoid
conjugates were a possible catabolite of cortisol in the in
vitro samples.
Since conjugated derivatives of cortisol
would tend to remain in the aqueous phase during extraction,
changes over time in the distribution of radioactivity in
the aqueous and organic phases should reflect changes in the
amount and type of steroidal components in the samples.
Therefore, 50 microliter aliquots of the following were
counted in scintillation cocktail:
extractions,
(1)
all samples before
(2) aqueous phase of all samples after
extractions, and (3) organic extracts reconstituted in 1 ml
methanol.
In addition, the tissue samples after extractions
were solubilized in ProtosolR and radioactivity measured in
order to account for all tritium added.
66
RESULTS AND DISCUSSION
Extractions:
In media incubated with liver tissue, the
amount of tritium in the organic phase decreased over time
concomitant with an increase in tritium remaining in the
aqueous phase.
Extraction of the liver tissue revealed that
extractable tritium was low in tissue at t=0, increased by
t=3, and decreased slightly over the next two samples
(Figure 13)
.
In the muscle tissue and media incubated with muscle,
after 24 hr most of the tritium was dichloromethane
extractable; very little remained in the aqueous phase or
the tissue itself after extractions.
In the incubated blank
(no tissue) and in the media control freshly prepared and
immediately frozen virtually all of the tritium was
extracted with dichloromethane.
HPLC:
Steroid moieties in media extracts which were
radioactive but did not co-elute with cortisol must be in
vitro metabolites of cortisol.
Exceptions to this were
radioactive steroidal peaks that were also evident in the
media blank incubated 24 hr without tissue.
Tentative
identification of steroidal components in the samples
followed co-elution with matching absorption characteristics
of a peak with one of the steroids in the standard cocktail.
Aside from the authentic cortisol included in the
media, a peak coeluted with cortisone, was radioactive, and
is thus tentatively presumed to be cortisone.
The peak was
67
Figure 13.
Abundance (uCi) of radio-labeled cortisol and
cortisol derivatives over time (1) extracted with
dichloromethane from incubation media (MEDIA),
from liver tissue (LIVER),
(2) extracted
(3) remaining in aqueous phase
after organic extraction (AQUEOUS), and (4) total
radioactivity accounted for (TOTAL) over 24 hr incubation.
68
0.08_
-0- MEDIA
-AI- LIVER
-0- AQUEOUS
TOTAL
..1'.1.1 -1 i 1 .1. i
0.00
0
3
6
9
12
15
TIME (HR)
18
21
24
69
only evident in the samples incubated 3 and 6 h.
A peak co-
eluting with cortisone was noted in the 24 h sample but it,
unlike cortisone, absorbed better at 280 nm than at 254 nm.
A third radioactive peak, evident only in the 24 h sample,
absorbed only at 280 nm and did not co-elute with any of the
standards in the cocktail.
70
CONCLUSIONS
If the changes in the distribution of radioactivity
evident over time in these samples do in fact reflect
formation of conjugated derivatives of cortisol, this in
vitro system may prove useful for characterizing the
relationship between clearance and cortisol dynamics
vivo.
in
The metabolism of cortisol to other steroids, while
evident in the system, is clearly not occurring in the same
magnitude as transformation to the putative steroid
conjugates.
This may in fact be an artifact of this system.
Kime (1978), for example, was able to demonstrate relatively
greater in vitro yields of cortisone and the chromatographic
technique he used was evidently sufficiently sensitive to
detect yields of 11-B-OH-AD and AT.
However, given the work
of Truscott (1979), I believe that in teleosts the liver,
and specifically hepatic catabolism of steroids to water
soluble conjugates, deserves careful study concerning the
role of clearance in driving circulating cortisol dynamics.