AN ABSTRACT OF THE THESIS OF
Mara L. Spencer for the degree of Master of Science in Fisheries Science presented on
November 17,2000. Title: Behavioral and Physiological Indicators of Stress in
Sablefish, Anoplopoma fimbria.
Abstract approved:
Redacted for Privacy
Carl B. Schreck
The development of assays for stress in marine fishes is vital for studying the
impacts of bycatch in fisheries and for determining the health of fish being cultured or
used in research. This research developed behavioral and physiological assays for
stress in juvenile sablefish, Anoplopoma fimbria, a species that comprises a valuable
North Pacific fishery and is often a substantial part of incidental discard. The effects of
conditions, intrinsic or extrinsic to the fish, on the variability of the stress response
were also investigated. A moderate stress of 15 minutes in air was used to elicit an
acute stress response. Behavioral responses and physiological values were evaluated at
1, 5, and 24 hours after the 15 minute air stress, and were compared with control fish
that received only a minimal air stress. In the first series of experiments, behavioral
patterns and changes in behavior over time of stressed and control fish were
determined, and the protocols and time course for measuring behavioral effects of
stress were established. In the second series of experiments, physiological assays were
added to the behavioral protocols developed during the first series of experiments.
The behavioral assays included activity levels, swimming speed at capture, and
appetitive behavioral patterns in response to a chemical food stimulus (squid extract),
and to a visual food stimulus (squid without odor). These behaviors are ecologically
relevant in terms of performance and survival. All of these behaviors were sensitive to
stress. In general, behavioral responses were depressed by stress at 1 hour, followed by
recovery to control levels by 5 hours. However, the intensity of behavioral responses
was affected by feeding history (1 or 5 days of food deprivation) and group influence
(recovering alone or in visual contact with 3 conspecifics), which therefore affected the
ability of the behavioral responses to assess stress. The behavioral assays were less
capable of detecting differences between stressed and control fish when the responses
of control fish were depressed as a consequence of being fed the day before. Visual
contact with conspecifics facilitated recovery of activity in stressed fish, but therefore
also resulted in apparent activity responses to chemical food stimulus that were more
likely attributable to activity increases of conspecifics than to appetitive behavior. The
focus of attention of isolated fish on activity of conspecifics often interfered with visual
detection of food.
The physiological assays included plasma concentrations of cortisol, glucose,
and lactate, all of which proved to be sensitive measures of stress in sablefish. These
parameters were elevated by stress at I hour, followed by a decreasing trend to 5 and
24 hours. The physiological assays were affected by feeding history, and an effect of
group influence was also indicated. Cortisol and lactate levels in stressed fish fed the
day before recovered faster than for stressed fish that were deprived of food for 5 days.
Glucose levels in stressed fish fed the day before were not elevated above controls.
These results suggested an alleviating effect of feeding on the biochemical stress
response. At 5 hours, cortisol and glucose were elevated above baseline levels in both
solitary stressed fish and in stressed fish influenced by a group, but also for controls
influenced by a group, suggesting an exacerbating effect of isolated fish being in visual
contact with groups. There were critical cortisol, glucose and lactate thresholds (180
ng/ml, 140 mg/dl, and 175 mg/dl, respectively) above which no appetitive behavioral
responses occurred. These clear demarcation levels are extremely valuable for linking
behavioral and physiological responses.
These results indicate that behavioral and physiological assays are sensitive
indicators of stress in sablefish, although the magnitude, time courses, and correlation
of responses may be affected by factors intrinsic and extrinsic to the fish that may vary
before and during recovery. There was a correspondence between behavioral and
physiological indications of stress shortly after the stressor had been removed and
levels of stress were still severe. However, there was a temporal discrepancy after
partial recovery had occurred, at which time recovery of physiological norms had not
yet been established although behavioral responses had recovered. While behavioral
patterns may readjust quickly, the persistence of an energetic load during recovery
from stress, as indicated by continued physiological perturbations, may compromise
ability to respond to additional stressors.
Behavioral and Physiological Indicators of Stress in Sablefish, Anoplopoma fimbria
by
Mara L. Spencer
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented November 17,2000
Commencement June 2001
Master of Science thesis of Mara L. Spencer presented on November 17, 2000
APPROVED:
Redacted for Privacy
Major Professor, representing Fisheries Science
Redacted for Privacy
Head of Department of Fisheries ancavildlife
Redacted for Privacy
D
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
ACKNOWLEDGEMENT
To all of my committee members I offer thanks for their willingness to share
their time, expertise and guidance. I sincerely thank my major advisor Carl Schreck for
his encouragement and advice, which were invaluable in guiding me throughout this
project. Bori Olla provided the impetus and support that made this work possible, and
his passion for behavioral research was a great inspiration. I am grateful for the
insightful ideas and knowledge that Martin Fitzpatrick and Frank Moore provided
during the process of experimental design and implementation, and for their interest
and time in being part of my committee.
Many people in the Oregon Cooperative Fish and Wildlife Research Unit
offered their assistance, answers to my questions, and reassurances for my
uncertainties. I would especially like to thank Beth Siddens for her support, and for her
patience and skill in teaching me how to run the biochemical assays and find my way
around the lab.
The people in the Fisheries Behavioral Ecology Program were all great sources
of encouragement and wisdom, and were instrumental and helpful in seeing me
through the entire process: Michael Davis, Michele Ottmar, Clifford Ryer, Susan
Sogard, Al Stoner, Erick Sturm, Cindy Sweitzer, and Richard Titgen.
A very special thanks goes to Wilfrido Contreras-Sanchez for his wonderful
help and encouragement in all aspects of this endeavor.
With deep appreciation I thank my mother, Nancy Engst, for sharing her
passion for the ocean and its inhabitants, and for sparking my interest in research.
Funding and research facilities were provided by the Fisheries Behavioral
Ecology Program of the Alaska Fisheries Science Center, National Marine Fisheries
Service at the Hatfield Marine Science Center, Newport, OR. Laboratory facilities
were also provided by the Department of Fisheries and Wildlife, Oregon Cooperative
Fish and Wildlife Research Unit at Oregon State University, Corvallis, OR.
TABLE OF CONTENTS
INTRODUCTION ........................... ................ .... ...................................................... 1
Sablefish ......................................................................................................... 2
The Stress Response ...................................................................................... 3
The Stressor ................................................................................................... 4
Assays ............................................................................................................ 5
Modifying Factors .......................................................................................... 9
Correspondence of Assays ............................................................................. 10
MATERIALS AND METHODS ................................................................................ 11
Sablefish Collection and Rearing Conditions ................................................. 11
Experimental Conditions ............................................................................... 11
Treatments ...................................................................................................... 14
Treatment 1: Food Deprivation .......................................................... 15
Treatment 2: Group Influence ............................................................ 15
Treatment 3: Absence of Food Stimulus ............................................ 16
Experimental Series 1: Behavioral Responses ............................................... 16
General Activity Patterns ................................................................... 17
Chemoreceptive Responses to Food .................................................. 20
Visual Responses to Food .................................................................. 21
Experimental Series 2: Behavioral and Physiological
Responses .......................................................................................... 22
General Activity Patterns, Chemoreceptive and
Visual Responses to Food ...................................................... 25
Swimming Speed ................................................................................ 25
Physiology Assays: Cortisol, Glucose, and
Lactate ..................................................................................... 26
Data Analysis ................................................................................................. 26
TABLE OF CONTENTS (Continued)
RESULTS ................................................................................................................... 28
Experimental Series 1: Behavioral Responses ............................................... 28
General Activity Patterns ................................................................... 28
Chemoreceptive and Visual Responses to Food ................................ 35
Experimental Series 2: Behavioral and Physiological
Responses .......................................................................................... 42
General Activity Patterns ................................................................... 42
Chemoreceptive and Visual Responses to Food ................................ 45
Swimming Speed ................................................................................ 48
Cortisol, Glucose, and Lactate ............................................................ 49
Assay Comparisons ............................................................................ 56
DISCUSSION ............................................................................................................. 64
Behavioral and Physiological Assay Comparisons ........................................ 64
Behavioral Responses ..................................................................................... 67
General Activity Patterns ................................................................... 68
Chemoreceptive and Visual Responses to Food ................................ 72
Swimming Speed ................................................................................ 77
Physiological Responses ................................................................................ 78
Cortisol ............................................................................................... 78
Glucose ............................................................................................... 81
Lactate ................................................................................................. 83
Concluding Remarks ...................................................................................... 84
REFERENCES ........................................................................................................... 85
APPENDIX ................................................................................................................ 99
LIST OF FIGURES
Figure Page
1. Diagram outlining the main characteristics of the
observation tanks for control (a) and stressed (b) fish .................................... 12
2. Experimental design for experimental series 1
(Behavior) ....................................................................................................... 18
3. Experimental design for experimental series 2
(Behavior and Physiology) .............................................................................. 23
4. Baseline patterns and effects of stress on mean activity
(± SE) for fish deprived of food for 1day (a) or 5 days
(b) in experimental series 1............................................................................. 29
5. Baseline patterns and effects of stress on mean activity
(± SE) for fish under solitary confinement (a) or in
visual contact with a group (b) in experimental series 1 ................................ 31
6. Baseline patterns and effects of stress on mean activity
(± SE) for fish experiencing no food stimulus in
experimental series 1 ...................................................................................... 34
7. Chemoreceptive response, expressed as activity index
(line Xlmin + biting occurrences) change (a) and visual
response, expressed as food consumption (b) in relation
to activity prior to the chemical and visual stimuli (xaxis) for fish in food deprivation (left) or group
influence (right) treatments of experimental series 1 ..................................... 37
8. Effects of stress on percentage of fish responding to
chemical extract (a, b) or visual food stimulus (c, d)
through time for fish in food deprivation (a, c) or group
influence (b, d) treatments and the linear relationship
between percentages of chemoreceptive and visual
responses for food deprivation (e) or group influence
(f) treatments of experimental series 1 ........................................................... 38
9. Baseline patterns and effects of stress on mean activity
(± SE) for fish deprived of food for 1 day (a) or 5 days
(b) in experimental series 2 ............................................................................. 43
LIST OF FIGURES (Continued)
Figure Page
10. Baseline patterns and effects of stres.s on mean activity
(± SE) for fish under solitary confinement (a) or in
visual contact with a group (b) for experimental series
2 ...................................................................................................................... 44
11. Effects of stress on percentage of fish responding to
chemical extract (a, b) or visual food stimulus (c, d),
and on mean swimming speeds (±SE) offish escaping
capture net (e, f) through time for fish in food
deprivation (a, c, e) or group influence (b, d, f)
treatments of experimental series 2 .............................................................. :.46
12. Baseline values and effects of stress on mean plasma
concentrations (± SE) of cortisol (a, b), glucose (c, d)
and lactate (e, f) for fish in food deprivation (a, c, e) or
group influence (b, d, f) treatments of experimental
series 2 ............................................................................................................ 50
13. Relationship between mean plasma concentrations (±
SE) of cortisol (a), glucose (b) and lactate (c) after 5
hours of recovery, and the number of days fish were
deprived of food in experimental series 2 ....................................................... 55
14. Physiological plasma concentrations and linear
relationships for individual fish in food deprivation (a,
c, e) or group influence (b, d, f) treatments of
experimental series 2. Relationships are between
glucose and cortisol (a, b), lactate and cortisol (c, d)
and lactate and glucose (e, f) ........................................................................... 57
15. Relationships between chemoreception responses and
plasma concentrations of cortisol (a, b), glucose (c, d)
and lactate (e, f) for the same fish in food deprivation
(a, c, e) or group influence (b, d, f) treatments of
experimental series 2 ...................................................................................... 60
16. Relationships between visual responses and plasma
concentrations of cortisol (a, b), glucose (c, d) and
lactate (e, f) for the same fish in food deprivation (a, c,
e) or group influence (b, d, f) treatments of
experimental series 2 ...................................................................................... 62
LIST OF APPENDIX FIGURES
Figure Page
1. Mean temperatures recorded during experimental
series 1 (a) and 2 (b), and relationship between weight
and total length for ~xperimental series 1 and 2 (c) ...................................... 100
2. Sample data sheet for recording of activity patterns for
experimental series 1 ..................................................................................... 101
3. Sample data sheet for recording of activity patterns for
experimental series 2 ..........';........................................................................... 102
BEHAVIORAL AND PHYSIOLOGICAL INDICATORS OF STRESS IN
SABLEFISH, ANOPLOPOMA FIMBRIA
INTRODUCTION
Developing an understanding of the capacity of marine fishes to respond to
stress is critical for assessing anthropogenic impacts and has important theoretical and
practical implications that are of potential benefit to researchers (Pickering 1981),
aquaculturists (Pickering 1981), and commercial fisheries managers (Chopin and
Arimoto 1995). Knowledge of what constitutes "normal" behavior and physiology
provides a basis for comparison to "stressed" animals, as well as indicating what
stressors may be important with respect to a particular species under particular external
and internal conditions, and what disruption of homeostasis may mean to the animal in
respect to survival and performance (e.g. Olla and Studholme 1975; and for reviews
see: Mayer et al. 1992; Schreck 1990). Within this context, pertinent assays can then
be developed which will indicate if an animal is stressed, describe the nature of the
stressor, and allow estimation of when or if the animal will recover from the stressor.
To take into account the dynamic nature of the stress response, several levels of
biological organization should be explored, as the stress response is an integration of
various levels (Adams 1990). Further, the plasticity of the stress response should be
investigated in order to understand how different external and internal variables of the
physiological, psychological and physical environment might modify the measurement
results (OIl a et al. 1974; Schreck 1981). This is useful for estimating the accuracy of
assays, explaining possible variations in measurements, and for pointing towards
possible methods for alleviating stress. Once various behavioral and physiological
measures are calibrated, then one or more measurements may be used to indicate
impacts on other behavioral and physiological functions (e.g. Jones et al. 1987).
The objectives of this research were to evaluate and compare nonlethal
behavioral and physiological assays for stress in juvenile sablefish, with regard to
sensitivity, efficacy, consistency, time course, and ecological relevance. An additional
objective was to evaluate the effects of altering the internal motivational state and the
2
external social context on the variability of the stress response as measured by these
assays.
Sablefish
Study of stress in sablefish provides valuable information for the commercial
fishery, ongoing laboratory research, and aquaculture development. Sablefish are an
important North Pacific commercial fishery both as a target species (McFarlane and
Beamish 1983a) and as bycatch (McFarlane and Beamish 1983b; Kendall and Matarese
1987). These fish are especially sensitive to impacts of overfishing due to their long
lives (McFarlane and Beamish 1983c), residence behavior (Beamish and McFarlane
1988), and dependence on strong year classes (McFarlane and Beamish 1983b, 1992).
Juvenile sablefish provide most of the recruitment potential for a fishery area rather
than movement of adults into an area (Beamish and McFarlane 1988; Kodolov 1983),
and can be a substantial amount of incidental discards (McFarlane and Beamish 1983c;
Sampson et al. 1997). The rapid growth ofjuvenile sablefish (Shenker and Olla 1986;
Kendall and Matarese 1987; McFarlane and Nagata 1987) may be particularly sensitive
to interruption of normal feeding as might occur under stressful conditions. Related to
their commercial value, sablefish have been studied under laboratory conditions to
provide information about their physiological (Sullivan and Smith 1982; Sullivan and
Somero 1983; Furnell 1987) and behavioral (L0kkeborg et al. 1995; Sogard and Olla
2000) ecology, and in the field to provide information about factors affecting their
biology and distribution (McFarlane and Beamish 1983b; Sasaki 1985; Grover and
Dlla 1986, 1987; Kendall and Matarese 1987). The feasibility of commercial
mariculture of sablefish is also being explored (McFarlane and Nagata 1987; Alderdice
et al. 1988; Solar et al. 1987, 1990; Clarke 1993; Whyte et al. 1994).
The ability to assess and understand the stress response in sablefish is useful for
evaluating and mitigating anthropogenic stressors incurred by fishing, research, and
culture conditions. To date, research on the impact of stressors in sablefish has
assessed the effects of tagging on mortality and maturation age (McFarlane and
Beamish 1990), the effects of hand-jigging and trapping on mortality (Rutecki and
3
Meyers 1992), and the effects of trawling, hooking, temperature and air stress on
mortality, blood biochemistry and feeding behavior (Olla et al. 1997, 1998; Davis et al.
2000). However, the characteristics of the stress response in sablefish have not been
addressed in detail.
The Stress Response
Stress is caused by physical, biological or chemical factors outside the normal
range of conditions that an animal experiences (for reviews of definitions of stress see:
Pickering 1981; Moberg 1985; Adams 1990; Wedemeyer et al. 1990; Barton and
Iwama 1991; Weiner 1992). The stress response results in changes in physiology and
behavior (Beitinger and McCauley 1990). These changes may affect survival and
performance capacity (Schreck 1981). Reduced performance capacity may ultimately
be manifested in growth, reproduction, and disease resistance (Adams 1990; Jobling
1994; Schreck 2000). Recovery from stress involves return of physiology and behavior
to pre-stress states. However, if the severity or duration of the stressor is beyond the
capacity of the animal to regain homeostasis mortality may result (Selye 1950, 1973;
Olla et al.1980; Schreck 2000).
The stress response is a dynamic and polymorphic process (Schreck 1981,
1990) that may impact all organizational levels of an animal as well as populations
(Shuter 1990), communities (Fausch et al. 1990), and ecosystems (Olla et al. 1980;
Wedemeyer et al. 1990) (see also: Adams 1990; Jobling 1994). It is difficult to clearly
separate stress response patterns into organizational or temporal units. This problem is
reflected in the variety of conflicting philosophies of the stress response (Weiner
1992). Hierarchical viewpoints are useful for organizational purposes, but do not
address the integration and complexity of the stress response. For example, when the
presence of a stressor has been processed neurally by an animal's sensory systems, the
first response may be behavioral (Slobodkin 1968), with subsequent physiological and
behavioral changes being integrated into the response if the stressor is not simply
avoided behaviorally (Olla et al. 1980). If the stressor is not avoided, then a series of
general responses is initiated.
4
Temporal stages of a generalized physiological stress response were described
by Hans Selye in 1950 as the General Adaptation Syndrome (GAS) concept in which
there are three stages, each with characteristic physiological events: the alarm reaction,
resistance, and exhaustion. The definition of the stress response was further developed
(Mazeaud et a1.l977; Wedemeyer and McLeay, 1981; Wedemeyer et a1.l990) to reflect
levels of biological organization involved in the stress response: the primary response
involves the neuroendocrine system and includes the release of stress hormones
(catecholamines and corticosteroids), the secondary response is the blood and tissue
alterations that result from primary level changes, and the tertiary response includes
changes in behavior, metabolic rate, disease resistance, growth rate, survival, and
reproduction (see also: Beitinger and McCauley 1990; Jobling 1994). Under this
definition of the stress response, behavior can be considered to be an integration of
physiological processes and the external and internal environments. While such
hierarchical conceptualizations are useful for explanatory purposes, they do not reflect
the polymorphic nature of the stress response. Mason (1968,1971, 1975a and b)
rejected the concept of a non-specific stress response as proposed by Selye (1950)
when he found that different stressors produce different hormonal responses, and some
stressors do not cause corticosteroid responses if the emotional stress component is
removed. A generalized physiological stress response, then, is elicited when there is a
psychological component of fear, discomfort, or pain to the stress (e.g. Schreck and
Lorz 1978; and for reviews see: Schreck 1981; Levine 1985; McEwen 1998).
Therefore, the physiological stress response is sensitive to psychological as well as
physical stimuli, both as components of the stress, and factors that may contribute to
alleviating or worsening the physical or psychological perception of stress.
The Stressor
The capacity of an animal to respond to stressors is best demonstrated by a
generalized stress response which is consistent and which will elicit measurable
changes in the range of behavioral and physiological responses possible (Olla et al.
1980; Beitinger and McCauley 1990; Schreck 1990). The severity, duration and nature
5
of the stress are factors that will affect the stress response. The stress must be within
the animals sensory realm, be recognized as stressful, and be within physiological and
behavioral ranges that can be responded to for a generalized response to be elicited
(Wedemeyer et al. 1990).
Handling is an acute stressor that has been shown to consistently produce a
generalized stress response within all biological levels of organization (e.g. Pickering
et al. 1982). The causal mechanisms of handling stress in fish are hypoxia, exertion,
and some form of psychological stress (e.g. Pickering et al. 1982; and for review see:
Schreck 1981). Handling stress is easily reproducible, lending itself well to research
use. Handling stress is an inevitable occurrence in laboratory and field research
(Pickering 1981), aquaculture (Pickering 1981), and fisheries processing (Chopin and
Arimoto 1995), all of which are current concerns for sablefish. Sensitivity to handling
stress can vary with species, ontogeny, reproductive status, and several other factors.
Assays allow assessment of handling stress under various biological and physical
conditions and indicate possible mitigative actions (Schreck 1990; Schreck et al. 1997).
Assays
Assays measure departures from behavioral and physiological norms
(Wedemeyer and McLeay 1981; Beitinger 1990; Schreck 1990). The most powerful
assays will be those that predict changes that are ecologically relevant for the animal
(Olla et a1.1980; Adams 1990; Barton and Iwama 1991). In addition to indicating the
severity and duration of the stress response, assays may also help to predict detrimental
consequences for the animal when subjected to stress. Factors in assay selection
include sensitivity, consistency, and ease of use (Heath 1990).
StUdying the integration of behavior and physiology is important in
understanding the capacity of animals to respond to stress (Schreck 1981; Adams 1990;
Heath 1990). Physiological parameters constrain and regulate behavior. Behavior
enables an animal's physiological state to interact with the environment (Beitinger
1990). Stressful conditions may be alleviated primarily by a behavioral response with
little detectable change in physiology (Olla et al. 1980; Moberg 1985), or primarily by
6
a physiological response with little detectable change in behavior (e.g. Rehnberg and
Schreck 1987). Alternatively, behavioral and physiological changes may be highly
integrated. A behavioral response to stress may be a result of physiological stimuli, or
alteration of behavior may produce the physiological changes needed to compensate
for the stress.
It is important to establish behavioral and physiological baselines of normality
in the study of the stress response. Establishing the normal ranges of behavioral (Olla
et a1.1980) and physiological (Schreck 1990; Mayer et al. 1992) parameters provides a
basis for comparison with stressed levels. From baselines of normality, predictions
may be made about which stressors will be important within the ecological context of
the animal. Also, these baselines will indicate what departures from the norm, both in
severity and duration, will mean in terms of the animal's ability to compensate (e.g.
Olla and Studholme 1975).
When a stress response is initiated there is an immediate endocrine cascade of
responses as well as an initial behavioral response mediated by the nervous system.
The endocrine response is coordinated in part by the hypothalamic-pituitary-interrenal
(HPI) axis, with the hypothalamus directly stimulating the release of catecholamines
and indirectly stimulating the release of corticosteroids (for reviews of endocrine
responses during stress see: Mazeaud et al. 1977; Donaldson 1981; Mazeaud and
Mazeaud 1981; Jobling 1994; Sumpter 1997; Wendelaar Bonga 1997).
Catecholamines and corticosteroids play vital roles in the subsequent cascade of events
in a generalized stress response. An advantage of measuring endocrine response
parameters is that they can rapidly indicate the presence and perhaps severity of stress.
The most widely used physiological stress response factor for a primary level
bioassay is plasma corticosteroid concentration. Cortisol is important in restoring
homeostasis and mobilizing energy reserves, especially during stress (Mommsen et al.
1999). Measurement techniques for plasma cortisol are well established, relatively
easy to perform, and typical responses are well known (Donaldson 1981). Most
research has focused on salmonids (Barton and Iwama 1991) although there is
accumulating evidence that similar plasma cortisol responses occur in many marine
fish (Thomas et al. 1981; Robertson et a1.1988; Waring et al. 1992, 1996; Barnett and
7
Pankhurst 1998; Tsunoda et al. 1999), including sablefish (Olla et al. 1997, 1998;
Davis et al. 2000), although exceptions exist (Vijayan and Moon 1994). A variety of
stressors have been shown to cause an increase in plasma cortisol concentration,
including handling (Barton and Iwama 1991). Peaks in plasma cortisol concentrations
often occur within minutes to hours, making a rapid assessment of the severity of stress
possible. Plasma cortisol concentrations can reflect cumulative levels of stress when
additional stressors are applied (e.g. Barton et al. 1986).
Levels of glucose and lactate in the blood are secondary stress response
measures which have been extensively used as bioassays of stress, primarily for
freshwater fish, especially salmonids, and data are accumulating for marine species
both for glucose (Robertson et al. 1988; Waring et al.1996) and lactate (Beamish 1968;
Wardle 1978; Waring et a1.l996). Typical glucose and lactate responses to stress have
been established for sablefish (Olla et al. 1998; Davis et al. 2000). Handling stress
produces changes in glucose and lactate concentrations in the blood that may be easily
sampled concurrently with cortisol. Hyperglycemia is initially induced by
catecholamine stimulation of glycogenolysis in the liver, and may then be maintained
by corticosteroid action. Hyperlacticemia in blood and muscle is due to anaerobic
metabolism caused by severe exercise and fright (Wedemeyer et al. 1990). Both
hyperglycemia and hyperlacticemia are initially adaptive responses that aid in
responding to stress, but may become maladaptive over time as energy reserves run
low as a consequence of increased metabolism and inanition, and homeostasis is no
longer possible to maintain. Blood glucose and lactate concentrations may be used as
indicators of energy debt caused by stress. Peaks in glucose and lactate concentrations
can occur rapidly and measure cumulative stress (e.g. Barton et al. 1986). As
secondary responses, elevations in glucose and lactate may be less sensitive to low
levels of stress that do not elicit a sufficient primary response to cause changes in
subsequent stress response events.
Tertiary stress response systems, including behavior, integrate primary and
secondary systems and are directly linked to whole animal consequences. They may be
used to predict population and ecosystem level consequences (Olla et al. 1980), and are
powerful tools for understanding stress (Schreck 1990). While they carry the extra
8
expenses of complexity and variability (Beitinger 1990), they have the ability to reflect
effects of stress long after other measures have returned to normal (Schreck 1990).
Behavior can be a very sensitive indicator of stress in fish and directly relevant
as to predictions of ecological consequences (Olla et al. 1980; Schreck et al. 1997).
Behavioral reactions to stress are critical in that they are the first line of defense
(Slobodkin 1968; Beitinger 1990) and can greatly alter the impact of the stress
(Moberg 1985; Olla et al. 1980). Behavioral assays may be used to measure
cumulative stress (e.g. Sigismondi and Weber 1988). Ecological relevance is critical to
the choice of a behavioral assay and requires knowledge of the life history and biology
of the species in question (Olla et al. 1974). Behavioral parameters may change
drastically with species, age, sex, nutritional status, condition, prior experience,
predation risk, and social interactions. The sensitivity of behavioral assays reflects the
importance of the particular behaviors to survival, growth, and reproduction. Ideally,
the behavioral measures used should be stable except under stressful conditions
(Beitinger 1990).
Feeding behavior is a well established indicator of stress in fish (Beitinger
1990). Changes in feeding behavior have consequences for energy available for
survival, growth, and reproduction. In general, stress causes a reduction or cessation of
feeding (Beitinger 1990). This may be especially critical for early life history stages
that are generally characterized by frequent feeding and fast growth (Beitinger 1990),
and are more susceptible to starvation (Love 1980). Towing in a simulated trawl has
been shown to affect feeding behavior in sablefish (Olla et al. 1997).
Feeding behavior is an integration of sensory systems and behavioral patterns
(Beitinger 1990), components of which may also be used to measure stress. Two
important sensory modalities for feeding behavior for pelagic fish are chemoreception
and vision. The general appetitive behavioral patterns (or phases) associated with
feeding are arousal, search, and ingestion (Beitinger 1990; Jones 1992). Sensory
systems contribute differentially to these phases, with the relative contribution
depending on species, age, feeding motivation, and environmental conditions (Atema
1980; Jones 1992). Stress decreases feeding motivation (Beitinger 1990). Appetitive
behaviors such as those associated with arousal and searching in response to various
9
food stimuli can measure changes in feeding motivation. The additional range of
sensitivity provided by measures of these behaviors will complement that of general
feeding behavior. Sablefish are chemoreceptively very sensitive and responsive to
food stimuli (L0kkeborg et al. 1995).
Modifying Factors
Factors that have the potential to contribute to the variability of the stress
response by altering the severity, time course or interaction of elements of the stress
response are those that can modify the impact of the stressor (Wendelaar Bonga 1997).
It is important to know which factors can influence the range of the stress response by
affecting either normal or stressed behavioral and physiological responses. In addition
to providing insight into the variability of the stress response, effects of modifying
factors will gauge the sensitivity of the assays being used. Modifying factors may
constrain the stress response by reducing response options, add to the stress response as
an additional stressor, or help to lessen the severity or duration of the stress response.
Due to interaction between various components affecting the stress response,
modifying factors may also cause disassociation between assays used to measure the
stress response. Modifying factors may be intrinsic or extrinsic to the animal (Schreck
1981; alla et al.1996). Intrinsic factors include: genetics, ontogeny, sex, reproductive
state, previous experience, and physiological state (Olla et al. 1974, 1996; Moberg
1985; Barton and Iwama 1991). Extrinsic factors may be physical, such as temperature
and light levels, or biological, such as social interaction and predation risk (Olla et al.
1974, 1996; Barton and Iwama 1991).
Feeding history (intrinsic) and social interactions (extrinsic) are two potentially
modifying factors that are variable given different research, fishing, and culture
conditions, and have the potential to affect behavioral and physiological parameters.
These factors may be altered to help alleviate stress and reduce time to recovery.
Juvenile sablefish are fast growing and gregarious, and consequently sensitive to
feeding- and social-related factors.
10
Correspondence of Assays
Comparisons of measures of stress from different levels of biological
integration enhance the ecological relevance of the particular measures, illustrate the
dynamics underlying the stress response, and permit comparison of the efficacy and
sensitivity of measures. Correspondence of measures from different levels of
biological organization enables inferences to be made as to the effects of a stress on
other parameters than the ones measured. Noncorrespondence may indicate temporal
or hierarchical differences in the roles that the measured parameters play in the stress
response, as well as which measures are more or less sensitive to a particular stress,
and which types of factors may interact with the stress response. Studies that combine
and compare primary, secondary, and tertiary measures of stress are limited for fish.
11
MATERIALS AND METHODS
Sablefish Collection and Rearing Conditions
Sablefish were collected in spring of 1996 and 1997 as young of the year (25-40
mm TL) about 40 km offshore of Newport, Oregon with the use of a neuston net. They
were reared in circular tanks (1.2 m diameter, 0.3 m depth, 339 1 volume) initially, and
then transferred into larger circular tanks (2.2 m diameter, 0.8 m depth, 30411 volume)
when they reached approximately 200 mm TL and at least 3 weeks prior to being used
in experiments. Rearing densities were 20 to 50 fish per tank. Rearing tanks were
supplied with sand-filtered and UV sterilized flow-through seawater (30-32%0 salinity,
O 2 > 90% saturation) at a replacement rate of 101 min-I. Seawater temperature ranged
from 9.5 to 14.8 °C with a monthly average of 11.7 °C in 1996-1997, and from 10.3 to
18.2 °C with a monthly average of 12.5 °C in 1997-1998. Fish were fed Oregon Moist
Pellets ® until they reached approximately 200 mm TL and then switched to a biweekly
feeding to satiation on chopped squid (Loligo opalescens) which was obtained from the
Depoe Bay Fish Company in Newport, Oregon, and stored at -15
0c.
Fish were
maintained on squid for at least 1 month prior to being used in experiments.
Experimental Conditions
The first series of experiments ran from October 1, 1996 to April 4, 1997 and the
second series ran from December 10, 1997 to March 8, 1998. Experiments were
conducted in two rectangular tanks (2.44 m length, 0.91 m height, 0.91 m depth) that
were divided into two equal sections by transparent Plexiglas (Fig. 1). One long wall
of each tank was made of transparent Plexiglas for viewing and videotaping. Each half
of the two tanks was visually separated into four equal-sized, vertically oriented
rectangular blocks with dark ribbon placed on the outside of the Plexiglas. Seawater
entered tanks at a total rate of 15 I min- I from two inputs in the bottom of each tank,
one in the center of each tank half. Seawater flowed out through four drains located at
the top of the tanks, two on each end. The flow rate was consistently maintained with
12
Figure 1. Diagram outlining the main characteristics of the observation tanks for
control (a) and stressed (b) fish. Lists on the right describe the treatments applied and
the responses measured.
•
•
•
Soaked Squid
Soaked Squid
Group (when present)
(a) Control
o
Water flow &
squid extract
Soaked Squid
Figure 1
Treatment 1: Food Deprivation
(Alone, Odor & Visual Assays)
J Day
5 Days
Treatment 2: Group Influence
(3 Days, Odor & Visual Assays)
Solitary
Visual Contact wit" Group
Treatment 3: No OdorNisual Assays
(Alone, 3 Days)
No Odor & No Visual Assays
Group (when present)
(b) Stressed
o
squid extract
Behavior
General Activity Patterns
Chemoreceptive Response
Visual Response
Swimming speed
Physiology
Cortisol
Glucose
Lactate
14
the use of valves and flow meters. The mean seawater temperature during the first
series of experiments was 11.0 ± 0.07 DC with a range of9.9 to 12.3 DC (Fig. la,
Appendix). In the second series of experiments the mean seawater temperature was
11.9 ± 0.02 DC and ranged from 11.2 to 12.4 DC (Fig. 1b, Appendix). Temperature was
not significantly different among treatments within either experimental series
(ANOVA, p < 0.05). The bottom of each tank was fitted with a sheet of perforated
gray PVC that allowed seawater to flow through uniformly throughout the tanks, and
did not allow food to fall through. Light was supplied by fluorescent lamps with
intensity of 10 mol m- 2s- l and heat energy of 5000 K which provided wavelengths that
simulated the light of the sun at noon (4870 K), and was similar to light levels of
holding tanks.
Subyearling fish were used in all trials. The mean length of fish in the first series
of experiments was 310 ± 5 mm with a range of 220 to 409 mm, and mean weight was
216.9 ± 10.7 g with a range of66.3 to 430.3 g. In the second series of experiments the
mean length was 324 ± 3 mm with a range of250 to 398 mm, and mean weight was
260.0 ± 7.8 g with a range of 113.8 to 535.5 g. Lengths and weights were not
significantly different among treatments within either experimental series (ANOVA, p
< 0.05) (Fig. Ic, Appendix).
Treatments
Each pair of fish, consisting of 1 control and 1 stressed fish, that were introduced
into the two experiment tanks was assigned to 1 of 3 treatments: 1) food deprivation, 2)
group influence, or 3) absence of food stimulus. These treatments were designed to
assess how the stress response may be altered by external and internal variables. In the
first series of experiments, all 3 treatments were conducted. The food deprivation- and
group influence-treatments were both carried out to the 48 hour observation period in
the first experimental series, but the food stimulus treatment was only carried out to the
5 hour period. In the second series of experiments only the first 2 treatments were
conducted. The food deprivation treatment was carried out to the 24 hr period in the
second experimental series, but the group influence treatment was only carried out to
15
the 5 hr period during these experiments. Treatments were alternately assigned in
order to control for time-related variability in factors such as fish size and seawater
temperature. See table in Appendix for a listing of treatments during each
experimental series and the number of fish assayed in each treatment.
Treatment 1: Food Deprivation
The purpose of the food deprivation treatment was to investigate the effects that
levels of feeding motivation had on the behavioral and physiological responses. In this
treatment fish were deprived of food for either 1 or 5 days before they were introduced
into the experiment tanks. Up until the designated time of food deprivation fish were
fed to satiation twice a week as described above. Fish were placed alone in the
experimental tanks, and received both the odor and visual food stimuli during the
behavioral observation periods.
Treatment 2: Group Influence
The group influence treatment was conducted to assess the influence of visual
contact with conspecifics on the behavioral and physiological responses. In this
treatment fish were either placed in the experimental tanks alone, or in visual contact
with 3 conspecifics of similar size that were intended to serve as facilitating groups.
The groups were introduced into the left sides of the experimental tanks 24 hours
before the control or stressed fish were introduced into the right sides of the tanks. The
facilitating groups were maintained in holding tanks of naive fish or fish that had been
used in a trial at least 3 weeks prior. All fish, including those in the groups, were
maintained on a biweekly feeding to satiation on squid and deprived of food for 3 days
prior to the beginning of a trial. In the first series of experiments the behaviors of the
experimental fish as well as those in the groups to which they were visually exposed
were quantified during the same observation periods. In the second series of
experiments only the behaviors of the isolated fish were quantified. During the
behavioral assays that were conducted to assess appetitive responses to the chemical
16
stimulus of food, the groups received the squid odor at the same time as the isolated
fish. For the behavioral assays conducted to assess appetitive responses to the visual
stimulus of food, each group was fed 3 pieces of soaked squid just before the isolated
fish on the other side of the tank was fed 1 piece of soaked squid.
Treatment 3: Absence ofFood Stimulus
This treatment was performed in order to investigate the changes in behavior over
the course of the first 6 hours after introduction of the fish into the experimental tanks
without the addition of either the chemical or visual food stimuli that were used in the
other treatments to assess appetitive behavioral responses. The data from this
experiment were used to establish the time course and recovery of normal activity
patterns of the control and stressed fish without the influence of food stimuli, and
determine if these normal activity changes could be misinterpreted as appetitive
responses. Fish were deprived of food for 3 days prior to introduction into the
experimental tanks, and were placed alone in the experimental tanks. These were the
same conditions as those for the fish in the group influence treatment that were placed
in the experimental tanks without visual contact with conspecifics, except for the
absence of food stimuli during the behavioral observation periods. Therefore, this
treatment served as a negative control for the treatment condition where fish were
alone and fasted for 3 days. Limited time and numbers of fish prevented negative
controls being conducted for more than one of the treatment conditions. This particular
treatment condition was selected as it represented the middle food deprivation level,
and activity patterns were not influenced by group behaviors. Trials were videotaped
for the entire 6 hours after introduction of fish into the experimental tanks.
Experimental Series 1: Behavioral Responses
Assay development was limited to behavioral responses in the first series of
experiments in order to establish behavioral baselines and the time course of recovery.
17
For this reason, observations. on behavior were more exhaustive in the first year. (See
Fig. 2 for a description of experimental design.)
For each trial, two fish were netted from one holding tanle Netting took less than
two minutes for each fish. The first fish that was caught was transported in a 20 I
bucket, half-filled with seawater, to the right side of one of the experimental tanks.
Transfer time took less than four minutes. This fish served as the control and it was
never out of water for more than a few seconds. In contrast, the second fish that was
caught was transferred to an empty plastic tub (34 x 24 x 13 cm) for 15 min before
being transferred to the right side of the other experimental tank. This fish was
designated as the "stressed" fish in reference to the long air stressor that it was
subjected to in addition to being transferred. Air temperature was 17 ± 1°C. The
location of the control and stressed fish was alternated between the two experimental
tanks to control for tank effects. Fish were not reused in experiments.
Each fish was videotaped for the first 2 hours after introduction into an
experimental tank, and subsequently at 4-6,23-25, and 47-49 hr after introduction.
Behavioral observations were conducted during each of these 2 hr intervals, which will
be referred to as the 1, 5, 24, and 48 hr observation times for convenience. In this first
series of experiments, general activity patterns, behavioral responses to chemical food
stimulus, and behavioral responses to visual food stimulus were evaluated as potential
behavioral assays.
General Activity Patterns
Activity patterns were determined by counting the number of lines that a fish
crossed per minute, and by determining the percent of time spent in each block per
minute. A Datamyte 800 data recorder (Electro General Corporation) was used to
perform these tasks. These data were recorded for each minute for the first 10 minutes
after a fish was introduced, for the 10 minutes just prior to food stimulus introduction,
for the 10 minutes after the chemical food stimulus was introduced, and for the 10
minutes after the visual and physical food stimulus. For all other intervals during the
two hours of videotaping, activity pattern data were recorded for every third minute
18
Figure 2. Experimental design for experimental series 1 (Behavior). The four
observation periods are labeled 1,5,24 and 48 hrs. For each observation period the
beginning and end of videotaping are indicated. Sample sizes for each observation
period are indicated in parentheses. Numbers in circles indicate introduction of squid
extract (1) or soaked squid (2) into experimental tanks. Descriptions of treatments are
summarized in text box.
19
T I ME
HR M I N
Experim ental Series 1: Behavior
OBSERY.
PER IOD
o
0
Start
Videotape
I Fish
I Fish
~
-----. ~
~
6
15 min
Air Stress
~
Group
o
o
15
50
20
5
24
48
CD
o
~
•1
Squid Extract ----. Chemoreceptive Response ---. Activity Changes
Soaked Squid ----. Vi sual Respon se
2
o
'dStOtP
VI eo ape
4
0
Start
Videotape
4
50
5
20
6
0
Stop
Videotape
23
0
Start
Videotape
23
50
24
20
25
0
47
o
47
50
48
20
49
o
Figure 2
ASSAYS
(n=7-9)
(n=7-9)
ffi
2
(End ofNo Odor/Visual Tmt.)
(n=7-9)
R,p'"
Repeat
Stop
Videotape
Stop
Videotape
Food Consumption
(No Odor/Visual TmL continue videotap ing until 6hr Omin)
R,p'"
Repeat
Start
Videotape
~
(n=5-7)
Repeat
Repeat
ffi
2
CD
CD
Tml. I) Food Deprivation
I Day
5 Days
(Solitary)
(With Odor/ Visual)
Tmt. 2) Group Influence
Solitary
3 Conspecifics
(3 Days)
(With Odor/ Visual)
Tmt. 3) No Food Stimulus
No Odor/ Visual
(Soli/my)
(3 Days)
20
(see Fig. 2 in Appendix for an example of a data sheet). For the absence of food
stimulus treatment that was videotaped from 0-6 hours, behavior data were recorded
during the same minutes of the I and 5 hour observation periods as just described, with
the addition of 10 min intervals each half hour in between the 1 and 5 hour periods.
The occurrence of biting at the tank bottom and at the feeding tube was also recorded.
For assessment of general activity patterns, data for each type of observation was
averaged over 10 minute intervals. Intervals of 10 minutes were chosen based on
preliminary analysis of activity patterns, and were intended to smooth out the minuteto-minute variation in activity patterns, while still allowing precise assessment of
activity changes over time. These data were used to assess differences in activity
levels, percent time spent in the different tank areas, and to develop protocols for the
chemoreceptive assay.
Chemoreceptive Responses to Food
At 50 min after the start of videotaping for a behavioral observation period each
fish received a chemical food stimulus. The chemical food stimulus consisted of squid
extract that was prepared as described in L0kkeborg et al. (1995). The extract was
stored in Whirl-Pacs
®
at -80°C and was brought out to thaw 25 min prior to use. In
each trial 20 ml of extract was injected by syringe through a valve in the seawater input
line. The injection point for each tank was located upstream from where the input lines
split to supply the two inputs to the bottom of each tank. Preliminary trials with red
food coloring determined that the flow of extract was uniform throughout the tanks and
that the residency time before being flushed out was approximately 1 hr. Response to
the extract was determined by comparing behavior before the extract introduction with
the behavior after. Preliminary analysis of changes in activity patterns in response to
the extract indicated that responses were immediate but transitory, typically lasting less
than 10 min. Consequently, 5 min directly before and directly following the extract
introduction were chosen as the appropriate time intervals to evaluate chemoreceptive
responses.
21
Preliminary results based on the extent and direction of the change in an activity
index (line crossings/min + occurrence of biting behavior) in response to food odor,
relative to the activity index level prior to the introduction of odor and visual food
stimuli, were used to develop a chemoreception response index where fish were
assigned a positive response when: 1) the pre-odor/visual food stimuli activity index
values were < 8 and there was an increase of greater than 1 unit/min in response to
squid extract; 2) the pre-odor/visual food stimuli activity index values were 2:8 and :::;12
and there was an increase or decrease of greater than 1 unit/min; or 3) the preodor/visual food stimuli activity index values were> 12 and there was a decrease of
greater than 1 unit/min. These criteria were established based on the three types of
activity responses that were observed when fish exhibited different levels of activity
prior to the introduction of the extract: When the activity index levels prior to extract
introduction were < 8 the activity of most of the fish increased in response to odor,
often by > 4 units; for activity index levels prior to extract introduction that were
between 8 and 12 there were both small increases and decreases in activity in response
to odor; and when activity prior to extract introduction was greater than 12, the
response to the odor stimulus was always a decrease in activity.
Visual Responses to Food
At 80 min after the start of videotaping for a behavioral observation period, a
visual and physical food stimulus was provided by the introduction of a piece of squid
mantle into the tan1e The squid used was the same as that being used for feeding fish
in the holding tanks. The squid pieces were approximately 2 x 2 cm square and 1 g ±
0.5 g in weight. To minimize the chemical aspect of the food stimulus, the squid
pieces were soaked in flowing seawater for at least one hour prior to use. The squid
was introduced through a PVC tube, which extended 15 cm beneath the water surface.
The tube was angled such that the opening above water extended back through a fabric
blind and the opening below the water surface was 15 cm from the tank wall. This
positioning allowed the movement of the squid through the water column to be the
22
only stimulus evident to the fish. Ingestion of the squid by the fish within 10 min was
the criterion for a positive response.
Experimental Series 2: Behavioral and Physiological Responses
The purpose of the second series of experiments was to compare behavioral and
physiological responses to stress, using the behavioral protocols established in the first
series of experiments. Based on the results of the first series of experiments,
behavioral and physiological assays were conducted at I, 5, and 24 observation times,
but not carried out at the 48 hr observation period. As the timing and protocols for
measurement of behavioral responses had been well established in the first series of
experiments, fewer behavioral data were taken in the second series. (See Fig. 3 for a
description of experimental design.)
As with the first experiment, two fish were netted from a holding tank for use in
each trial. The control fish was transferred directly into the right side of an
experimental tank, and the stress fish was kept in air for 15 min before being
transferred into the right side of the other tank. Control and stressed fish were
alternated between the two experimental tanks and fish were not reused. Videotaping
began immediately before fish were introduced into the experimental tanks. Each fish
was videotaped for the first 2 hours after introduction into an experimental tank, and
subsequently at 4-6 and 23-25 hours after introduction. For convenience, the
behavioral and physiological assays conducted during these intervals will be referred to
as occurring at the 1, 5, or 24 hr observation periods. Additionally, because it was
noted in the first experimental series that general activity patterns of stressed fish
usually recovered sometime between the I and 5 hr observation periods, fish in the
second experimental series were videotaped during the interval between the 1 and 5 hr
periods to better characterize recovery of general activity patterns.
Unlike the first experimental series, on 12 different days (from 1II 0/98 to 3/8/98)
spread throughout the second experimental series, an additional fish was netted out of
the same holding tank immediately before the two treatment fish were netted out. Of
the 12 fish selected, 5 had been deprived of food for 1 day, 2 for 3 days, and 5 for 5
23
Figure 3. Experimental design for experimental series 2 (Behavior and Physiology).
The three observation periods are labeled 1, 5, and 24 hrs. For each observation period
the beginning and end of videotaping are indicated. Sample sizes for each observation
period are indicated in parentheses. Numbers in circles indicate introduction of squid
extract (1) or soaked squid into (2) experimental tanks, and removal of fish from
experimental tanks (3). Descriptions of experiments are summarized in text box.
24
TIME
HR M I N
OBSE RV.
PE RI OD
o
Ex perim ental Se ries 2:
Behavior & Phys iology
H oldi ng
Tank
1,3,5 d Food
Deprived
0
Stal1
Videotape
1 Fish
2 Fish
,;
-I-------~
------. ~
1 Fish
~
,4j_ _ _.~:mfi; mI1l!i1i1lfi
Group
. . . . ;. . . . .
0::J
Base lin e
Phys iology
(n = 12)
15 m in
"
~
Air Stress
~iiiiiiI.,...-1Ifi
Group
o
0
~BO'hFi~
15
50
20
o
CD
ASSAYS
(Tmt. 1 n= 15-1 6; Tmt. 2 n=6)
Soaked S qu id
40
2
•
•
Squid Extracl - . Chemoreception Response ---. Act ivity Changes
~ Vi sual Response
(2) Nel & Anestheti::e
~
Blood Pl asma
O R--'
0
(Continue Vid eotap in g)
5
24
(Tmt. I n=9- 10; Tmt. 2 n=6)
4
0
4
50
5
20
5
40
6
0
Stop
Videotape
23
0
Start
Vid eotape
23
50
24
20
24
40
24
45
Figure 3
Stop
Videotape
•
OR --.
~ Food Consumpti on
R,p'"
Repeat
~
2
Repeat
3
(End o/Tml. 2)
(Tmt. I n=2 -4)
R,p'"
Repeat
~2
Repeat
3
~
Swim Speed
C0I1 iso i
Glu cose
Lactate
Tml. I) Food Deprivation
I Day
5 Days
Behavior. 0-2,4-6,23 -25 h
Physiology.' 0-2, 4-6, 23 -25 h
(Solitary)
Tml. 2) Group Influence
Solitary
3 COl1specijics
Behavior: 0-2, 4-6 h
Physiology. 4-6 h
(3 Days)
25
days. These fish were immediately anesthetized and blood sampled, and the samples
were used to establish resting levels of cortisol, glucose, and lactate from unstressed
fish with a range of sizes, holding tank conditions and fasting levels that corresponded
to those of the fish being used in the experiments.
General Activity Patterns, Chemoreceptive and Visual Responses to Food
General activity patterns were determined similarly as in the first experimental
series. As in the first series, activity data were recorded for the 10 min after a fish was
introduced into an experimental tank, for the 10 min just prior to the chemical extract
introduction, for the 10 min after the chemical extract was introduced, and for the 10
min after the visual food stimulus was introduced. However, unlike the first series,
activity data were not recorded every 3 minutes for the intervals between these 10 min
observation periods. (See Fig. 3 in Appendix for an example of a data sheet.) While
only one of the treatments in the first series of experiments was videotaped for the
entire first 6 hr after fish were introduced into the experimental tanks, all of the
treatments in the second series were videotaped for the first 6 hr. Data were thus
recorded for 10 min at half hour intervals between the 1 and 5 hr observation periods
for all the treatments in the second series. The chemoreceptive and visual appetitive
assays were conducted exactly as in the first experiment.
Swimming Speed
Swimming speed of fish immediately before being captured and removed from an
experiment tank for blood sampling was an additional behavioral assay in the second
experimental series. The path of each fish was digitized with a transparent digitizer
system (Scriptel Corporation) for 10 sec starting from the instant the capture nets
entered the water. The average speed was recorded in mm/sec.
26
Physiological Assays: Cortisol, Glucose, and Lactate
Sampling for physiological assays, which were to be compared with the
corresponding behavioral assays, took place 20 min after the introduction of soaked
squid mantle during the last behavioral assay period for a pair of control and stressed
fish. For example, if a pair of control and stressed fish was to be sampled for
physiology at the 5 hr period, then the behavioral assays were run at the 1 and 5 hr
periods as described above. Twenty minutes after the soaked squid was introduced
during the 5 hr period both fish were then removed for blood sampling. After the
blood sampling the fish was returned to a recovery tank and not reused.
Two nets were used to capture the fish from the experimental tank. Capture time
took less than 3 min. The fish were anesthetized with 200 ppm buffered tricaine
methane sulfonate. Blood was taken from the caudal vein with 3 ml potassium EDTA
vacutainers. Plasma was immediately frozen and stored at -80°C. Plasma assays were
performed in the Oregon Cooperative Fish and Wildlife Research Unit laboratory at
Oregon State University. Plasma cortisol concentrations were determined by
radioimmunoassay as described by Foster and Dunn (1974), as modified by Redding et
al. (1984), and as validated for sablefish by Olla et al. (1997). Plasma glucose
concentrations were determined using the colorimetric procedure of Wedemeyer and
Yasutake (1977). Plasma lactate concentrations were assayed with fluorimetry
(Passonneau 1974).
Data Analysis
Individual fish were treated as the experimental unit. All behavioral and
physiological responses were analyzed using SASTM, release 7.0 (SAS Institute Inc.,
Cary NC, USA). A repeated measures analysis of variance (repeated ANOVA; GLM
procedure) on the activity patterns, measured as line crossings/minute and averaged
over 10 minute intervals, was performed for each observation period to determine
significant differences between activity patterns of control and stressed fish, and to
determine significant changes in activity over time for stressed fish in visual contact
27
with groups during the 1 hour observation period. Analyses of percentage
chemoreception and visual responses were conducted using the Fisher's exact test
(FREQ procedure), and pairwise comparisons were performed using the same test
when significant differences were indicated. The analysis of swimming speed, cortisol,
glucose, and lactate data over time was performed using a two-way analysis of variance
(ANOV A; GLM procedure) with treatment and time as variables, and multiple
comparisons were performed using a Tukey-Kramer adjustment. These data were
further analyzed with a two-way analysis of variance at each observation period to
determine interactions between stress level and levels of food deprivation and group
influence. All correlation values and linear relationship models between physiological
measurements were obtained using a linear regression analysis (REG procedure). Data
from the 48 hr observation period ofthe first series of experiments was not included in
statistical analyses.
28
RESULTS
Experimental Series 1: Behavioral Responses
General Activity Patterns
Activity in stressed fish was minimal during the 1 hour observation period, but
had increased to control levels by the 5 hour observation period. However, whether the
depressed activity of the stressed fish during the 1 hour observation period was
detectably less than the activity of control fish depended on the treatment. Activity
levels of fish often increased following food stimulus introduction. This was especially
apparent after the odor introduction, which provided a preliminary indication that an
analysis of response to food odor based on changes in activity was feasible.
Length of food deprivation affected the activity levels of control fish, and
therefore whether the activity patterns were detectably different between control and
stressed fish during the 1 hour observation period (Fig. 4). Activity of control fish
deprived of food for 5 days was significantly greater than that of control fish deprived
of food for 1 day during the 1, 5, and 24 hour observation periods (p = 0.0050, 0.0195,
0.0485 respectively). Activity patterns of stressed fish deprived of food for 1 or 5 days
were not different at any time period. There was not a significant difference between
control and stressed fish at the 1 hour observation period for fish fed the day before (p
= 0.0661) due to the low activity levels of the control fish as well as the stressed fish
during this observation period (Fig. 4a). However, for fish that were deprived of food
for 5 days there was a significant difference in activity between stressed and control
fish at the 1 hour period (p = 0.0004) with the control fish having consistently greater
activity than the stressed fish that exhibited minimal activity throughout the
observation period (Fig. 4b). There were no significant differences between control
and stressed fish within either of the food deprivation treatments during subsequent
observation periods. Although mean activity levels of stressed fish that were deprived
of food for 1 day appeared to be consistently above those of the corresponding controls
throughout the 24 hour observation period, the difference was not significant (p =
t
12 ~~~---------------r========~--~================================~--------------II----I
~ Co ntrol
-0- Stress
(a) 1 Day 10
Squid Odor Introd uced
'-V Sq uid Piece Introduced
8
6
,-...
.:
E
4
2
~ 0
Q)
c
(b) 5 Days
'-'
.f' 10
tT
.~
.....
~ 8
6
4
2
o
min
hr
p=0.0004
15
0
25
40
55
75
"1 hr" 95
115
S
4
25
40
5S
7S
"5 hr"
95
115
5
23
25
40
55
75
"24 hr"
95
115
15
47
25
40 55
75
95
115
"48 hr"
Figure 4. Baseline patterns and effects of stress on mean activity (± SE) for fish deprived offood for Iday (a) or 5 days (b) in
experimental series 1. Arrows indicate introduction of squid extract (black) or soaked squid (gray) . The timeline (x-axis)
indicates the hour that videotaping began for an observation period, and minutes after initiation of videotaping during an
observation period. Each of the four observation periods is two hours in length and is designated as the 1,5, 24 or 48 hr
observation period. P-values are shown whenever significant differences were found between the control and stressed
treatments at a particular observation period. Sample sizes are 7 for the 1, 5, and 24 hr observation periods and 5-6 for the 48 hr
observation period.
30
0.0942). Activity patterns were extremely variable for all treatments during the 48
hour observation period.
Presence of conspecifics influenced the activity patterns of the experimental
fish by decreasing the variability in activity patterns in general, and by increasing the
activity of the stressed fish during the 1 hour observation period (Fig. 5). For fish that
were alone there was a significant difference between control and stressed fish at 1
hour (p = 0.0377) with the stressed fish displaying minimal activity throughout the
observation period (Fig.5a). However, fish that were in visual contact with
conspecifics did not exhibit differences between control and stressed fish at 1 hour
(Fig.5b). In this case the stressed fish began increasing activity approximately 1 hour
after introduction into the observation tank, reaching the level of activity being
exhibited by the controls during the 1 hour observation, and then exceeding the activity
of the controls during the 5 hour observation period (p = 0.0183). The increase in
activity during the 1 hour observation period of the stressed fish in visual contact with
conspecifics appeared to coincide with the time that the squid odor was introduced.
When the first 5 activity means (activity prior to extract) of the 1 hour observation
period for stressed fish in visual contact with groups were each compared with the
following 6 means (activity after extract), 3 of the means post-extract were greater than
all of the 5 pre-extract means (p < 0.05) and 4 of the post-extract means were greater
than 4 of the pre-extract means (p < 0.05). There were no significant differences
between solitary control and stressed fish after the 1 hour period, and there were no
significant differences between control and stressed fish within view of groups after the
5 hour period. Activity patterns for control fish alone and with groups, and for stressed
fish alone and with groups, were not different during any observation period. Activity
increases following introduction of food stimulus were consistently strong, preceded by
relatively low activity levels, and followed by a quick return to the preceding lower
levels for all observation periods for control fish within view of groups, and for the 5,
24, and 48 hour periods for stressed fish within view of groups. Activity patterns for
both control and stressed fish within view of groups closely followed the activity
patterns offish in groups, especially during the 5, 24, and 48 hour periods (Fig. 5c).
Variation in activity patterns between replicates was very low for the facilitating
31
Figure 5. Baseline patterns and effects of stress on mean activity (± SE) for fish under
solitary confinement (a) or in visual contact with a group (b) in experimental series 1.
Mean activity patterns (± SE) are also shown for the neighboring groups (c). Arrows
indicate introduction of squid extract (black) or soaked squid (gray). The timeline (xaxis) indicates the hour that videotaping began for an observation period, and minutes
after initiation of videotaping during an observation period. Each of the four
observation periods is two hours in length and is designated as the 1,5,24 or 48 hr
observation period. P-values are shown whenever significant differences were found
between the control and stressed treatments at a particular observation period. Sample
sizes are 7 for the 1, 5, and 24 hr observation periods and 5-6 for the 48 hr observation
period.
12 ~-------------------------------------r=================================='-------------------~
-e-
(a) Solitary
10
~ Squid Odor Introduced
Co ntrol
-0- S tress
W Squid Piece Introduced
8
6
4
2
o
,.-..
::
E
X
p=O.0377
-e-
(b) With Group
10
Co ntrol
-0- Stress
8
p=O.0183
(c) Groups
10
8
6
4
2
o
min
hr
5
0
Figure 5
25
40
55
75
"1 hr"
95
25
4
40
55
75
"5 hr"
95
5
23
25
40
55
75
"24 hr"
95
115
5
47
25
40
55
75
"48 hr"
95
115
w
N
33
groups throughout all observation periods, and was correspondingly low in the control
and stressed fish within view of the groups, compared with other treatments, especially
during the 24 and 48 hour observation periods.
Activity patterns of fish in the treatment where food stimuli were not
introduced indicated that overall activity levels and the effects of stress on activity
were similar to the other treatments that did receive food stimuli, and provided
supporting evidence that the brief increases in activity following the introduction of
food stimuli noted in the other treatments were associated with appetitive behavior
rather than a normal background variation in activity (Fig. 6). Activity levels of
stressed fish were significantly lower than that of controls during the 1 hour
observation period (p = 0.0049). Activity patterns of the control and stressed fish
converged between 2 and 4 hours after the fish were introduced into the experimental
tanks such that they were no longer different during this time period, and they
remained similar during the 5 hour observation period. A comparison of activity levels
between control fish with no food stimuli with the corresponding control fish that were
also solitary and deprived of food for 3 days but did receive odor and visual food
stimuli indicated no differences between controls during the 1 and 5 hour observation
periods. Likewise, the stressed fish that did not receive odor and visual food stimuli
during the 1 and 5 hour observation periods were not different from the stressed fish
that did. No increases in activity of the magnitude noted after food stimuli introduction
in the other treatments were apparent in the activity patterns of the fish that did not
receive odor and visual food stimuli.
The percentage of time spent in the upper and lower portions of the tanks was
affected by stress and by level of food deprivation. Time spent in the left or right
portions of the tanks was affected by group presence but not by stress. Over all of the
treatments, fish spent most of their time during the 1,5, and 24 hour observation
periods in the lower half of the tanks (92.5%, p = 0.0000), and more time in the left
half (56.2%), which contained the feeding tube, than in the right half (43.8%) (p =
0.0088). Percentage of time spent in different tank areas did not change significantly
for any of the treatments in relation to the introduction of either the odor or the visual
food stimulus.
12 ~----------------------------------------------------------------------~
i'
10
.S
8
::
~
'-"
....s;:
."
:;:
c:.>
-<
-+- Control
- 0 - Stress
6
~
:§
No OdorNisual Food Stimuli
4
2
0
p=0.0049
min
hr
5
25
40
55
75
95
115
30
60
2
0
II
1 br"
90
5
25
40
55
75
95
115
4
"3 br"
"5 br"
Figure 6. Baseline patterns and effects of stress on mean activity (± SE) for fish experiencing no food stimulus in
experimental series 1. Bars under x-axis indicate the time at which introduction of squid extract (black) or soaked squid
(gray) occurred in other treatments but did not occur in this treatment. The timeline (x-axis) indicates the hour that
observations began for a particular observation period, and minutes after initiation of observations during an observation
period. Each of the three observation periods is two hours in length and is designated as the 1,3, or 5 hr observation period.
The vertical dotted lines visually separate the 1 and 5 hr observation periods that correspond to the 1 and 5 hr observation
periods of the treatments that did receive food stimulus. P-values are shown whenever significant differences were found
between the control and stressed treatments at a particular observation period (n=8).
35
The effect of stress on the amount of time spent in different areas of the tanks
was only apparent in the vertical use of tank space during the 1 hour observation period
of the food deprivation treatment. The level of food deprivation influenced the amount
of time control fish spent in the upper half of the tanks, and therefore the magnitude of
differences between control and stressed fish. Stressed fish, deprived of food for 1 or 5
days, spent little time in the upper portion of the tanks, 0.46% ± 0.28 and 0.42% ± 0.27,
respectively. However, control fish that were deprived of food for 1 day also spent
little time in the upper portions of the tanks (1.08% ± 0.75), and were therefore not
different from stressed fish deprived of food for 1 day. Control fish deprived of food
for 5 days spent significantly more time (14.59% ± 5.10) in the upper half of the tanks
during the 1 hour observation period than the control fish deprived of food for 1 day (p
= 0.0058), and the stressed fish deprived of food for 5 days (p = 0.0037). Over all of
the observation periods, control fish that were deprived of food for 5 days spent a
significantly greater percentage of time (16.2 % ± 5.7) in the upper half of the tanks
than did the controls deprived of food for 1 day (1.4% ± 0.6) (p = 0.0404). Stressed
fish that were deprived of food for 1 and 5 days spent comparatively moderate amounts
of time in the upper half of the tanks, 2.4% ± 1.3 and 7.9% ± 4.4 respectively, and were
not significantly different from the control fish that were deprived of food for 1 or 5
days.
The social attraction of fish within visual contact of a facilitating group was
apparent in the greater (p < 0.0001) amount of time both the control (89.3% ± 3.3) and
stressed (81.9% ± 5.5) fish spent on the left (group) side of the tanks over all of the
observation periods compared to both the control (42.9% ± 9.9) and stressed (44.3% ±
8.4) fish that were alone. There were no significant differences between control and
stressed fish, either alone or with a group, during any of the observation periods.
Chemoreceptive and Visual Responses to Food
The responses of the fish to chemical and visual food stimuli supported the use
of the chemoreception response index that was developed in this study in order to
account for individual variation in activity levels and response patterns. When the
36
activity index (line crossings/min + occurrence of biting behavior) prior to the
introduction of odor or visual food stimulus was greater than 12 units, the response to
the odor stimulus was a decrease in activity in all cases for both the food deprivation
and group influence treatments (Fig. 7a). For activity index levels prior to food
stimulus that were between 8 and 12 units, there were both small increases and
decreases in activity (within a range of ± 4 units) in response to odor. The activity of
most ofthe fish increased in response to odor, often by > 4 units, when the activity
index levels prior to food stimulus were < 8. All fish with activity index levels
~
8
prior to food stimulus responded to the visual food stimulus by immediately consuming
the introduced piece of squid (Fig. 7b). The chemoreceptive index that was developed
incorporated these three types of responses.
Stress generally depressed chemoreceptive and visual responses to food during
the 1 hour observation period, with recovery to control levels by the 5 hour period (Fig.
8). However, as with general activity levels, whether chemical and visual responses of
control and stressed fish were detectably different during the 1 hour observation period
depended on the level of food deprivation and the presence of conspecifics.
As with the general activity levels, 1 day of food deprivation depressed the
responses of controls to squid odor during the 1 hour observation period such that there
was not a difference between control and stressed fish, even though the responses of
the stressed fish were minimal (Fig. 8a). Consequently, while there was a clear
difference among the treatments during the 1 hour observation period (p
=
0.0023), the
responses of control and stressed fish were detectably different only for the fish
deprived of food for 5 days (p = 0.0047), with 100% of the control fish responding and
only 14% of the stressed fish responding. Fish deprived of food for 1 day did not show
a difference between control and stressed fish due to the low percentage (43%) of
control fish that responded to squid odor compared with the also low percentage (14%)
of stressed fish that responded. There were no significant differences among fish
deprived of food for 1 and 5 days in percentages of fish responding to squid odor
during the 5,24, and 48 hour observation periods.
Also similar to the general activity levels, the presence of conspecifics resulted
in an increase in the responses of stressed fish to squid odor during the 1 hour
Food Deprivation
16
~
=
-=U
ell
0::
~
~
"0
=
.>....
:~
....
CJ
-<
12
8
4
0
Group Influence
'V
•••
yi'q
,.
O~.'V ~
~ 'V • • ~
..
-~~-.----~----------------
o•
-4
-8
•
-12
-16
= YES
o
:g,
e
=
=
U
'"
o
-g o
r..
NO
~
o ••
..
•
2
4
6
'V
•
1 Day - C
1 Day - S
5 Days - C
'V 5 Days - S
•
•
8
10
12
14
16
18
20
•
'V
22
o
2
4
6
8
10
12
14
•
Solitary - C
Solitary - S
Social- C
Social- S
o
0
(b) Visual Response
o
0000.
16
18
20
22
Pre-OdorNisual Food Stimuli Activity Index
Figure 7. Chemoreceptive response, expressed as activity index (line Xlmin + biting occurrences) change (a) and visual
response, expressed as food consumption (b) in relation to activity prior to the chemical and visual stimuli (x-axis) for fish in
food deprivation (left) or group influence (right) treatments of experimental series 1. Horizontal dashed line demarcates where
values indicate no changes in activity patterns in response to chemical extract. Sample sizes are 21 for food deprivation and 2227 for group influence.
38
Figure 8. Effects of stress on percentage of fish responding to chemical extract (a, b)
or visual food stimulus (c, d) through time for fish in food deprivation (a, c) or group
influence (b, d) treatments and the linear relationship between percentages of
chemoreceptive and visual responses for food deprivation (e) or group influence (f)
treatments of experimental series 1. Common letters next to values in a particular
observation period indicate values that are not significantly different from each other
within that period. For observation periods where letters do not appear next to values
there were no significant differences found between any of the treatments. For both the
chemoreception and visual responses, sample sizes are 7 at observation periods 1,5,
and 24, and 5-6 at the 48 hr period (food deprivation), and 7-9 at observation periods 1,
5, and 24, and 5-7 at the 48 hr period (group influence). Sample sizes are 7-9 for the
linear relationship between responses.
39
(a)
..
,-,
Food Deprivation
100
a\
~
'-'
~
c
80
c..
~ 60
~
~ 40
~
CJ
~
~
e
20
~
.c
U
(C)
..
,-,
~
b)'
1 Day-C
·0 1 Day - S
-....- 5 Days- C
-'e'- 5 Days - S
0
'"0c
60
'"
c.:::
40
c..
~
-;
=
'"
~
20
0
_
Solitary-C
Solitary - S
-....- Social - C
-'e'- Social - S
·0
b •
(d)
100
80
a,b
_
>
a~~
'-'
~
.~
0
• a,b
a,b
~
0
~
/
~~..
0
c.:::
Group Influence
(b)
"v
.j
........
b •
48
24
5
24
5
48
Time (hr)
(e)
(f)
100
..
,-,
~
'-'
80
'"c0
60
'"
c.:::
40
=
20
~
c..
~
-;
'"
~
\7
•
•
0
0
•
•
~
~
r2=0.59
r2=0.84
0
0
0
0
20
40
60
80
100
0
20
Chemoreceptive Response (%)
Figure 8
40
60
80
100
40
observation period compared with stressed fish that were alone (Fig. 8b). Thus, while
there were significant differences among the percentages of fish responding to squid
odor during the 1 hour observation periods (p = 0.0226), only the solitary fish exhibited
a significant difference between control and stressed fish (p = 0.0294) during the 1
hour period, with 56% of controls responding to the squid odor as opposed to 0% of the
stressed fish responding. Stressed fish within view of groups did not differ from
control fish, in this case because a relatively high proportion of stressed fish (38%)
changed their activity patterns after the introduction of squid odor compared with
control fish (63%).
A small percentage of control fish in the treatment that did not receive odor
stimulus during the observation periods falsely indicated a chemoreceptive response
based on the activity index used. During the 1 hour observation period 13% of the
controls indicated a response, and 25% indicated a response during the 5 hour
observation period. None of the stressed fish at either observation period indicated a
response. There was no difference between control and stressed fish at either period.
Similar to the chemoreceptive and general activity responses, 1 day of food
deprivation depressed the responses of controls to the visual stimulus of food during
the 1 hour observation period such that there was not a difference between control and
stressed fish, even though the responses of the stressed fish were minimal (Fig. 8c).
Thus, although there was a clear difference among treatments in percentages of fish
responding to the visual stimulus during the 1 hour observation period (p = 0.0004),
differences between control and stressed fish during this period were only significant
(p = 0.0291) for the fish deprived of food for 5 days, with 86% of control fish
responding to the visual stimulus compared with 14% of the stressed fish responding.
Only 43% of the control fish that were deprived of food for 1 day responded to the
visual stimulus at the 1 hour period. Consequently, although none of the stressed fish
deprived of food for 1 day responded to the visual stimulus at the 1 hour period, the
differences between control and stressed fish that were deprived of food for 1 day were
not significant due to the small number of replicates along with the low number of
control fish responding. There were no significant differences among fish deprived of
41
food for 1 and 5 days in the percentages offish visually responding during the 5, 24,
and 48 hour observation periods.
As with the general activity and chemoreceptive responses, the presence of
conspecifics resulted in the visual appetitive responses of controls to not be different
from the responses of stressed fish during the 1 hour observation period (Fig. 8d).
However, unlike the general activity and chemoreceptive responses, this was due to
treatment effects on the responses of control fish rather than on responses of stressed
fish. Similar to the chemoreceptive response, there were differences among treatments
in the percentages of fish responding to the visual stimulus of food during the 1 hour
observation period (p = 0.0387). During the 1 hour observation period, a greater
percentage of solitary control fish (67%) responded to the visual stimulus of food than
did solitary stressed fish (11 %) (p = 0.0498), and control and stressed fish in visual
contact with groups were not significantly different. In contrast with the
chemoreceptive response, this lack of difference between control and stressed fish in
visual contact with groups was due to a low number of controls responding (50%)
compared with stressed (12.5%), rather than a high number of stressed fish responding.
There were no significant differences between numbers of solitary fish and fish within
visual contact of groups responding to visual food stimulus at the 5, 24, and 48 hour
observation periods.
Percentages of chemoreceptive and visual response were correlated for the food
deprivation and group influence treatments. For the food deprivation treatment there
was a strong correlation between the chemoreceptive and visual responses (R2 = 0.84)
(Fig. 8e). However, chemoreceptive and visual responses were less correlated for the
group influence treatment (R2 = 0.59) (Fig. 8f). In many cases a lower percentage of
control and stressed fish recovering within view of groups responded to the visual
stimulus of food than responded to the odor stimulus.
42
Experimental Series 2: Behavioral and Physiological Responses
General Activity Patterns
In the second series of experiments, which added physiological responses to the
behavioral protocols that were established in the first series, activity was depressed by
stress during the 1 hour observation period, with recovery to control levels by the 5
hour period, similar to the results of the first series. However, some of the differences
in activity that were related to the effects of the food deprivation and group influence
treatments that were noted in the first series of experiments were not evident in the
second series.
There was minimal activity during the 1 hour observation period in stressed fish
that were deprived of food for 1 or 5 days, followed by recovery of activity to control
levels by the 5 hour observation period (Fig. 9). These results were similar to those of
the first series of experiments. In contrast, the activity of the control fish that were
deprived of food for 1 day were not depressed compared with controls that were
deprived of food for 5 days, and therefore both control fish deprived of food for 1 day
and control fish deprived of food for 5 days were more active during the 1 hour
observation period than stressed fish that were deprived of food for 1 and 5 days (p <
0.000 I). Activity patterns of control fish deprived of food for 1 day and control fish
deprived of food for 5 days were not significantly different during any observation
period. Likewise, activity patterns of stressed fish deprived of food for 1 day and
stressed fish deprived of food for 5 days were not different at any observation period.
As in the first series of experiments, the stressed fish recovering alone in the
second series of experiments exhibited minimal activity throughout the 1 hour
observation period, while activity in the stressed fish recovering with groups began to
be evident following the introduction of squid odor during the 1 hour observation
period (Fig. 10). The activity of stressed fish that were solitary and stressed fish that
were with groups recovered to control levels by the 5 hour observation period. In
contrast to the first series of experiments, the presence of activity during the 1 hour
observation period of the stressed fish recovering with groups did not result in the
12
(a)
10
8
t Squid Odor Introdu ced
-+-
1 Day
Co ntr ol
--0- Stress
t Squid Piece Introduced
'V
n=3
n=9
n= 15
6
-a
4
,.-.,
~
::s
2
c
, .
0
X
~
.S
'--'
-
;;..,
.",::
.~
I
(b)
10
<.J
<
5 Days
n= IO
n= 16
8
11=4
n=1O
t
6
4
2
0
min
hr
p=<O.OOOI
15
0
25
45
65
"1 hr"
85
30
105
2
60
"3 hr"
90
5
4
25
45
65
"5 hr"
25
85
23
45
65
85
105
"24 hr"
Figure 9. Baseline patterns and effects of stress on mean activity (± SE) for fish deprived of food for 1 day (a) or 5 days (b) in
experimental series 2. Arrows indicate introduction of squid extract (black) or soaked squid (gray). The timeli.ne (x-axis) indicates
the hour that observations began for a particular observation period, and minutes after initiation of observations during an
observation period. Each of the four observation periods is two hours i.111ength and is designated as the 1, 3,5 or 24 hr observation
period. The vertical dotted lines visually separate the 1, 3 and 5 hr observation periods. P-values are shown whenever significant
differences were found between the control and stressed treatments at a particular observation period.
12
WSquid Odor Introduced
- . - Control
- 0 - Stress
10
'¥ Squid Piece Introduced
8
6
-
4
---:::
2
E
0
Q.l
.S
X
Q.l
(b YWith Group
C
::::.,
10
::
u
8
£;;.
6
~
4
2
0
p=O.OOIO
min
hr
5
0
25
45
65
"1 hr"
85
30
105
2
60
"3 hr"
90
5
4
25
45
65
85
105
"5 hr"
Figure 1O. Baseline patterns and effects of stress on mean activity (± SE) for fish under solitary confinement (a) or in
visual contact with a group (b) for experinlental series 2. Arrows indicate introduction of squid extract (black) or soaked
squid (gray). The timeline (x-axis) indicates the hour that observations began for a particular observation period, and
minutes after initiation of observations during an observation period. Each of the four observation periods is two hours in
length and is designated as the 1, 3, or 5 hr 0 bservation period. The vertical dotted lines visually separate the 1, 3 and 5 hr
observation periods. P-values are shown whenever significant differences were found between the control and stressed
treatments at a particular observation period (n=6-7).
45
differences between stressed and controls being undetectable, as the activity of the
controls was relatively high in the second series of experiments. Control fish that were
solitary were significantly more active during the 1 hour observation period than
stressed fish that were solitary (p = 0.0051) (Fig. lOa). Control fish within visual
contact of groups were significantly more active during the 1 hour observation period
than stressed fish within visual contact of groups (p = 0.0010) (Fig. lOb). Activity
patterns of control fish that were solitary were not different from control fish with
groups during any of the observation periods. Activity patterns of stressed fish that
were solitary were not different from stressed fish with groups during any of the
observation periods.
Chemoreceptive and Visual Responses to Food
Stress had a similar effect on the chemoreceptive and visual responses to food
in the second series of experiments as it did in the first, with both responses being
depressed at the 1 hour observation period for stressed fish, followed by recovery by
the 5 hour observation period. While levels of food deprivation and the presence of
conspecifics affected both chemoreceptive and visual responses to food in the first
series of experiments, these treatments only affected the chemoreceptive responses in
the second series of experiments.
One day of food deprivation depressed the responses of control fish to squid
odor during the 1 hour observation period such that there was not a detectable
difference between control and stressed fish, even though the responses of the stressed
fish were minimal. As a result, the significant differences among treatments during the
1 hour observation period (p = 0.0033) were limited to the responses of control and
stressed fish deprived of food for 5 days (p = 0.0152), with 83% of the control fish and
0% of the stressed fish responding (Fig. IIa). Control and stressed fish deprived of
food for 1 day did not differ due to the low response (33%) of the control fish
compared with the stressed fish (0%).
The presence of conspecifics increased the responses of stressed fish to squid
odor during the 1 hour observation period such that a difference between responses of
46
Figure 11. Effects of stress on percentage of fish responding to chemical extract (a, b)
or visual food stimulus (c, d), and on mean swimming speeds (± SE) offish escaping
capture net (e, f) through time for fish in food deprivation (a, c, e) or group influence
(b, d, f) treatments of experimental series 2. For the chemoreceptive and visual
responses, common letters next to values in a particular observation period indicate
values that are not significantly different from each other within that period. For
observation periods where letters do not appear next to values there were no significant
differences found between any of the treatments. For the swimming speeds, values that
are not different between treatments within a particular observation period share
common letters, and values that are not different between observation periods within a
treatment share common numbers. Sample sizes are 6-7 for the 0, 1, and 5 hr sampling
periods and 2-4 for the 24 hr period.
Figure 11
48
control and stressed fish was not detectable (Fig. 11 b). While there were significant
treatment differences among percentages of fish responding to squid odor at 1 hour (p
= 0.0005), differences between control and stressed fish were only detectable (p =
0.0152) for solitary fish, with 83% of controls responding compared to 0% of the
stressed fish responding. Control and stressed fish that were within view of groups
were not significantly different at 1 hour. In this case, although 100% of the control
fish responded, a high percentage of stressed fish (43%) also had a response to squid
odor (or to the groups' increases in activity).
In contrast to the chemoreceptive responses, a high percentage of both controls
deprived of food for 1 and 5 days responded to visual stimulus of food at the 1 hour
observation period (Fig. llc). Consequently, the significant differences among
treatments (p < 0.0001) observed at the 1 hour observation period for the visual
responses to food included significant differences between control and stressed fish for
both 1 and 5 day food deprivation treatments. The percentages of both the controls
deprived of food for 1 day (83%) and the controls deprived of food for 5 days (100%)
that visually responded were greater than the response (0%) in both the stressed fish
deprived of food for 1 and 5 days (p = 0.0152 and 0.0022, respectively).
The percentages of fish responding to the visual food stimulus during the 1 hour
observation period were also not affected by the presence of conspecifics (Fig. lId).
Thus, the significant differences among treatments at the 1 hour observation period (p
< 0.0001) included differences between both control and stressed fish that were solitary
and fish that were with conspecifics. A greater percentage of control fish that were
solitary (83%) responded to the visual food stimulus than stressed fish that were
solitary (0%) (p = 0.0152), and a greater percentage of control fish within view of
groups responded (100%) than stressed fish within view of groups (14%) (p = 0.0047).
Swimming Speed
Stress seemed to decrease the swimming speed of fish being chased by a net
during the 1 hour observation period, with subsequent recovery to control levels by the
5 hour period. However, 1 day of food deprivation also depressed swimming speed at
49
the 1 hour observation period (Fig. lIe). As a result, at the 1 hour observation period
the swimming speeds of control fish that were deprived of food for 1 day were not
significantly faster than those of stressed fish deprived of food for 1 day, although
control fish deprived of food for 5 days were significantly faster than stressed fish
deprived of food for 5 days (p = 0.0330). Swimming speeds of control fish deprived of
food for 1 and 5 days did not change significantly with time. Swimming speeds of
stressed fish deprived of food for 1 and 5 days increased significantly with time.
Swimming speeds of escaping fish were only recorded at the 5 hour observation period
for the group influence treatments, and did not differ among any of the treatments at
this time (Fig. 11 f).
Cortisol, Glucose, and Lactate
In general, cortisol, glucose and lactate levels were elevated at the 1 hour
observation period in response to the 15 min air stress, had decreased by the 5 hour
period, and had fully recovered by the 24 hour period to the levels of control fish and
to the baseline levels of undisturbed fish that were sampled directly from holding tanks
(Fig. 12). In most cases, cortisol and glucose of control fish were also elevated midway between levels of holding tank fish and stressed fish at the 1 hour observation
period due to the stress of transfer into the experimental tanks. Cortisol levels of
control fish decreased to baseline levels by the 5 hour period. For control fish with
elevated glucose levels, recovery to baseline levels had occurred by the 24 hour period.
Level of food deprivation affected the timing of recovery of cortisol and lactate levels
towards baseline levels in stressed fish and affected the magnitude of the glucose
responses. An effect of presence of conspecifics was indicated by differences in levels
of cortisol and glucose elevations in control and stressed fish at the 5 hour observation
period compared to baseline levels of fish from holding tanks.
At the 1 hour observation period, cortisol levels in stressed fish deprived of
food for 1 and 5 days were similar and elevated above controls deprived of food for 1
and 5 days (p < 0.0001 and < 0.0016, respectively), and cortisol levels in all fish,
including controls, were elevated above the baseline values of fish in holding tanks (p
50
Figure 12. Baseline values and effects of stress on mean plasma concentrations (± SE)
of cortisol (a, b), glucose (c, d) and lactate (e, f) for fish in food deprivation (a, c, e) or
group influence (b, d, f) treatments of experimental series 2. Asterisks indicate values
that are different from baseline values of fish from holding tanks. Values that are not
different between treatments within a particular observation period share common
letters, and values that are not different between observation periods within a treatment
share common numbers. Sample sizes are 6-7 for the 0, 1, and 5 hr sampling periods
and 2-4 for the 24 hr period.
51
250
225
200
c
E
Oil
c
'-'
'0
io
~
U
Food Deprivation
(a)
*a
175
l~.*.a3
*b 6
150
~
125
*b 8
100
75
•
(b) Group Influence
•
Holding Tank
_
Solitary - C
• O· Solitary - S
~ Social-C
-'i't- Social - S
•
Holding Tank
_
IDay-C
. O· 1 Day - S
~ 5 Days - C
5D.y,.S
... 14 -"'*a
,.
"-...
, . a,b 7
50
b 9
25
--- --- --.
"-...
• Ii
o ~-,-.---------,--------~------~~
(d)
(c)
175
150
.-.
125
E
100
~
'-'
*a~
~
oCol
~
=
G
75
50
•
25
o ~-.~r---------'-------~~------~--250
(e)
(f)
225
200
.-.
175
--
150
~
E
';'
('iI
Col
('iI
125
100
...:l
o
Figure 12
1
5
24
Time (hr)
•
o
beyb
5
52
< 0.0001) (Fig. 12a). However, whether the stressed fish had recovered to baseline
levels at the 5 hour period depended on the level of food deprivation. While all
treatments significantly reduced their cortisol levels from 1 to 5 hours (p < 0.05),
cortisol levels of stressed fish deprived of food for 5 days were still elevated at the 5
hour observation period relative to the baseline levels of fish from holding tanks (p <
0.0001), whereas the levels for the rest of the treatments had decreased sufficiently as
to be no longer different from baseline values. Cortisol levels in stressed fish deprived
of food for 5 days further decreased from the 5 to the 24 hour observation periods (p =
0.0320), and were no longer elevated above baseline levels. There were no changes in
cortisol levels from the 5 to the 24 hour observation periods in the rest of the
treatments. All but the solitary control fish of the group influence treatments at the 5
hour observation period still had elevated cortisol levels compared with the baseline
levels offish in the holding tanks (p < 0.05) (Fig. 12b).
The effect of stress on glucose levels at the 1 hour observation period was
different depending on the level of food deprivation (p < 0.0001) (Fig. 12c). At the 1
hour period, level of food deprivation affected whether glucose levels of controls were
elevated above baseline levels of fish in holding tanks and whether glucose levels of
stressed fish were elevated above control levels. At the 1 hour observation period
glucose levels of all of the food deprivation treatments except for those of the controls
deprived of food for 1 day were significantly.elevated compared with the baseline
cortisol levels obtained from undisturbed fish sampled in the holding tanks (p <
0.0001). Stressed fish deprived of food for 5 days had the highest levels of glucose at
the 1 hour period and were significantly greater than all of the other treatments at 1
hour (p < 0.0001). In contrast, glucose levels of stressed fish deprived of food for 1
day were not significantly elevated above glucose levels of controls deprived of food
for 1 day. Glucose levels of control fish deprived of food for 5 days were elevated
above those of controls deprived of food for 1 day at the 1 hour period (p = 0.0106).
Glucose levels of all treatments except the 1 day deprived of food controls were still
elevated at the 5 hour observation period relative to the baseline levels of fish in
holding tanks (p < 0.05). Glucose levels of the stressed fish deprived of food for 5
days at the 5 hour observation period had decreased significantly from the 1 hour levels
53
(p < 0.0001) but were still significantly elevated compared with all ofthe other
treatments (p < 0.01). There were no other treatment differences during the 5 hour
observation period. Glucose levels of stressed fish deprived of food for 5 days further
decreased from the 5 to the 24 hour observation periods (p < 0.0001). Glucose levels
of stressed fish deprived of food for 1 day also decreased over time, but only
significantly from the 1 to the 24 hour observation periods (p = 0.0057). Glucose
levels of control fish deprived of food for 1 day did not change over time, whereas
glucose levels of control fish deprived of food for 5 days decreased significantly from
the 1 to the 24 hour observation periods (p = 0.0341). All treatments had recovered to
baseline holding tank levels at the 24 hour observation periods and there were no
significant differences between treatments at this time. As with cortisol, all but the
solitary control fish of the group influence treatments at the 5 hour observation period
still had elevated glucose levels compared with the baseline holding tank levels (p <
0.05) (Fig. 12d).
As with cortisol, lactate levels in stressed fish deprived of food for 1 and 5 days
were similar at the 1 hour observation period and were elevated above both baseline (p
< 0.0001) and control values (p < 0.0001) (Fig. 12e). At the 5 hour observation period
the recovery of lactate levels of stressed fish towards baseline values was different
depending on the level of food deprivation (p
= 0.0061). Unlike cortisol, lactate levels
were not elevated above baseline values for control fish at the 1 hour observation
period. Lactate levels of control fish deprived of food for 1 and 5 days were not
significantly different from each other, or baseline levels, at any of the observation
periods. Lactate levels of both stressed fish deprived of food for I and 5 days
decreased significantly from the 1 to the 5 hour observation periods (p < 0.0001), but
were still significantly elevated over baseline (p < 0.0001) and control levels (p <
0.0001). However, lactate levels in stressed fish deprived of food for 1 day had
decreased significantly more than the levels in stressed fish deprived of food for 5 days
at the 5 hour observation period (p
= 0.0023). Lactate levels further decreased from the
5 to the 24 hour observation periods in both stressed fish deprived of food for I and 5
days (p < 0.0001), and were no longer elevated above baseline or control levels. There
54
were no changes in lactate levels over time for control fish deprived of food for either 1
or 5 days.
In contrast to cortisol and glucose, there were no differences in lactate levels at
the 5 hour observation period that were related to the presence or absence of
conspecifics (Fig. 12f). Lactate levels of solitary stressed fish and stressed fish within
view of groups at the 5 hour observation period were still significantly elevated in
comparison to controls and baseline levels (p < 0.0001). There was no difference in
lactate levels at the 5 hour period between stressed fish recovering alone or with
groups. Lactate levels of the control fish that were alone and with groups were not
different from each other or from baseline levels.
Cortisol, glucose and lactate levels of stressed fish at the 5 hour observation
period increased significantly with the number of days that fish were deprived of food,
but did not increase for control fish (Fig. 13). Cortisol levels in stressed fish increased
significantly from 1 to 5 days of food deprivation (p = 0.0070) (Fig. 13a). Glucose
levels were also significantly greater in stressed fish deprived of food for 5 days than in
stressed fish deprived of food for 1 day (p = 0.0004) (Fig. 13b). In addition, glucose
levels in stressed fish deprived of food for 3 days were significantly greater than levels
in stressed fish deprived of food for 1 day (p = 0.0195). Lactate levels in stressed fish
increased significantly from 1 to 5 days of food deprivation (p
= 0.0154) (Fig. 13c).
Differences of cortisol and glucose levels between control and stressed fish increased
with the number of days of food deprivation. Cortisol levels were not significantly
different between control and stressed fish deprived of food for 1 and 3 days, but were
different for fish deprived of food for 5 days (p = 0.0194). Glucose levels were not
significantly different between control and stressed fish deprived of food for 1 day, but
were different for 3 days (p = 0.0456), and even more so for 5 days (p = 0.0158).
Lactate levels were significantly different between control and stressed fish at all levels
of food deprivation (p < 0.0001).
55
150
125
(a)
_
2
a
Control
-0- Stress
"0
... 50
o
a
.~
100
U
3
25
3
8
3
a
o ~---r------------------'-----------~----~-----
150
125
--t 100
e
'-'
Q.j
oCool
'"
:=
G
(b)
2
8
2
a
75
~=--+----7
Lb
50
8 =
3
3
8
3
25
o ~--~------------------,-----------------~-----
150
125
25
(c)
b
3
a
1,2
'8
b
3
b
2
3
01-~·!=============~·~============~·~-1
3
5
Days of Food Deprivation
Figure 13. Relationship between mean plasma concentrations (± SE) of cortisol (a),
glucose (b) and lactate (c) after 5 hours of recovery, and the number of days fish were
deprived of food in experimental series 2. Values for control and stressed treatments
that are not different at a particular number of days of food deprivation share common
letters, and values that are not different between days of food deprivation within control
or stressed treatments share common numbers (n = 6).
56
Assay Comparisons
Regressions of the blood parameters showed variation in correspondence of
assays based on their sensitivities to stress and to the treatments. There was an overall
positive relationship between the physiological parameters. Linear regression was the
model that best fit the data. Correspondence between assays was better for stress
treatments than for controls. Level of food deprivation strongly affected the
correspondence of assays. Group influence did not seem to affect correspondence of
assays, although there were not enough data points for accurate assessment. Cortisol
and glucose were more sensitive to stress than lactate. Glucose was the most sensitive
measure to feeding history.
In general, as cortisol increased, glucose increased. However, the food
deprivation treatments greatly affected this correspondence (Fig. 14a). Cortisol and
glucose of the stressed fish deprived of food for 5 days had the best correspondence (R2
= 0.89), followed by control fish deprived of food for 5 days (R2 = 0.48), and then by
stressed fish deprived of food for 1 day (R2 = 0.45). There was no correspondence
between cortisol and glucose levels for control fish deprived of food for 1 day, as
glucose levels remained low regardless of cortisol levels in this treatment. Thus, the
correspondence between cortisol and glucose was better when fish were deprived of
food for 5 days or stressed. As cortisol increased, there was a corresponding increase
(R2 = 0.84) in lactate only if fish were stressed (Fig. 14c). Lack of correspondence for
control fish was also evident for the relationship between glucose and lactate (Fig.
14e). Lactate increased for stressed fish but not for controls. Because of the depressed
glucose response in stressed fish deprived of food for 1 day, there was less of a
correspondence between glucose and lactate for fish deprived of food for 1 day (R2 =
0.62) than for fish deprived of food for 5 days (R2 = 0.82). While data for assessing the
correspondence among blood parameters for the group influence treatments were
limited, the lack of sensitivity of lactate levels to the transfer stress of control fish,
compared with glucose and lactate, was apparent in these treatments as well (Fig. 14f).
57
Figure 14. Physiological plasma concentrations and linear relationships for individual
fish in food deprivation (a, c, e) or group influence (b, d, f) treatments of experimental
series 2. Relationships are between glucose and cortisol (a, b), lactate and cortisol (c,
d) and lactate and glucose (e, f). Where associations were different between
treatments, separate regression lines are shown. For the group influence treatments
most models resulted in no significant correlations (regression lines not shown).
58
180
•o
160
,.-..
~
e
'-'
~
ell
o
~
G
Food Deprivation (a) 1 Day- C
1 Day - S
5 Days - C
5 Days - S
•
\7
140
.
•
•
\7
"
o
•
••••
.. .C? ......0
~
00 R=0.45
' b•. R2=0.00
"
y=O.llx+50.85
~
.
\7
2
R =0.42
y=O.42x+56.03
y=0.Olx+53.94
~~----~----'-----r----'-----.----~
50
o
300
.
Solitary - C
Solitary - S
Social- C
Social - S
\7
~
\7.
R2=0.48
~ \7 _ y=0.48x+56.41
f!:}._
60
20
100
200
250
300
Cortisol (ng/ml)
150
(c) 250
R2=0.84
y=0.78x+28.12
,.-.. 200
.....CIS
R2=0.89
\7 /
y=0.4lx+57.06 .. ~ \7\7
~ . --.~ ~
.-0h .• ....
•....•
80
40
~
.!
•
o
\7.
120
100
Group Influence
(b)
o
100
50
150
(d)
o
o
150
~
't 100
CIS
...;l
50
~
o
o
300
....CIS
100
50
R 2=O.62
y=3.92x-I31.73
o
:=:: 200
t
••
~-.".~
o
150
0
o
50
100
150
(f)
o
o
\7
0
0\7
~
....-- ..
o
R2=0.33
y=0.09x+3.86
200
250
300
Cortisol (ng/ml)
150
(e) 250
.!
....
o
o
W
't
100
CIS
\7
\7
R2=0.82
y= 1.89x-77.09
o
\7
o
0
...;l
50
2
R =O.01
y=O.05x+8.20
o
20
Figure 14
40
60
80
100
120
140
160
180 40
Glucose (mg/dl)
•
60
80
100
120
140
59
There were clear concentrations of physiological variables above which no
appetitive behavioral responses occurred (Figs. 15 and 16). These cutoffs for
behavioral responses were maintained regardless of treatment, and were essentially the
same for both chemoreceptive and visual food responses. These critical values were:
180 ng/ml for cortisol, 140 mg/dl for glucose, and 175 mg/dl for lactate. Below these
threshold values appetitive responses were variable and affected by the treatments.
Physiological responses for the group influence treatment were only determined at 5
hours, therefore peak values for physiological parameters at 1 hour were not available
to indicate the presence of cutoff values for behavior. Similar to appetitive behavior,
general activity prior to the introduction of food stimulus was minimal
«
1 line
crossing/min in all cases) when concentrations of the physiological variables were
above the threshold values, and was variable and not correlated with concentrations of
the physiological variables when values were below the thresholds.
60
Figure 15. Relationships between chemoreception responses and plasma
concentrations of cortisol (a, b), glucose (c, d) and lactate (e, f) for the same fish in
food deprivation (a, c, e) or group influence (b, d, f) treatments of experimental series
2. On the y-axis "yes" indicates fish that responded to squid extract and "no" indicates
fish that did not respond.
61
Food Deprivation
(a)
(b)
Group Influence
~
'co"
c.
~ Yes
==c
-
.~
c~.
y
o~ No
e
•...
~
..c
U
0
\1
•• ....
I
\10"'0
1 Day - C
1 Day - S
S Days - C
S Days - S
~ \1~V
0
<!.
•...
\1
100
200
250
300
Cortisol (ng/ml)
150
(C)
w
0
Solitary - C
Solitary - S
Social- C
Social- S
0
50
0
\1
50
0
100
150
(d)
~
'co"
c.
~ Yes
c
o
;
c.
~
y
~
t:;
e
\1
No
\liJ
W
\1
~ 0
0
~
..c
U
~
20
40
60
80
100
120
140 160 180 40
Glucose (mg/dl)
80
60
100
120
140
(t)
(e)
~
'"C
o
c.
~ Yes
-
==c
"I "
0
0
8
•
W\1
0
\1\1\1 0
\1 CD \1
.~
c~.
y
o~ No
o
e
~
..c
U
o
Figure 15
50
100
150
200
250
300
Lactate (mg/dl)
o
50
100
150
200
62
Figure 16. Relationships between visual responses and plasma concentrations of
cortisol (a, b), glucose (c, d) and lactate (e, f) for the same fish in food deprivation (a, c,
e) or group influence (b, d, f) treatments of experimental series 2. On the y-axis "yes"
indicates fish that responded to the visual food stimulus and "no" indicates fish that did
not respond.
63
Food Deprivation
(a)
c:o
~
c.
(b)
Group Influence
Yes
~
=
:; No
-;
~
CD
•
..
\1
tP
\1~V
0
\1
50
0
\1
100
200
150
o
~
40
W
0 ~O<D
60
80
100
~
c.
120
W
140
ocB
Yes
300
50
0
100
150
(d)
\1
160
180 40
Glucose (mg/dl)
(e)
c:
o
250
Cortisol (ng/ml)
-;
=
Solitary - C
Solitary - S
Social- C
Social- S
0
(C)
:; No
"Y ..
•.
I Day - C
I Day - S
5 Days - C
5 Days - S
0
20
0
V\l\1 \1
60
80
100
120
140
(f)
..
0
cIS 0\1
\1W
<D \1
~
~
-;
=
~
:; No
•
o
Figure 16
00
50
100
150
200
250
300
Lactate (mg/dl)
o
50
100
150
200
64
DISCUSSION
Behavioral and Physiological Assay Comparisons
For sablefish in this study, there were biochemical values (cortisol = 180 ng/ml,
glucose
=
140 mg/dl, lactate = 175mg/dl) above which no appetitive behaviors
occurred, providing inference capabilities for the assessment of the effects of stress on
behaviors vital to fitness, based on established physiological parameters. Once these
critical values were established, subsequent biochemical samples with values greater
than these thresholds would have strongly predicted that feeding behavior would also
have been impaired. However, below these thresholds feeding behavior was not
predictable from biochemical values. Although behavioral responses to stress often
have direct pertinence for survival or performance, behavioral assays are often difficult
to perform. If the underlying physiology of behavioral responses can be determined,
then physiological measures can be used to infer behavioral outcomes. As an example,
in a study by Strange et al. (1978) the return of normal feeding behavior corresponded
with the return of cortisol to basal levels during acclimation of chinook salmon
(Oncorhynchus tshawytscha) to continuous confinement stress. In this case,
subsequent cortisol measurements could have been used to infer the return of normal
feeding behavior.
While sablefish behavior was profoundly affected by stress when biochemical
values were above the threshold values, below these thresholds behavior was variable
and dependent on treatments. Jones et al. (1987) discuss a similar phenomenon in
Arctic char (Salve linus a/pinus). For control and recovered char, there was no
correlation between physiological and behavioral measures. However, for acidstressed char there were several physiological and behavioral correlations, suggesting a
threshold above which stress strongly affected behavior. Activity was correlated with
protein and glucose in acid-stressed char, and five blood parameters, including cortisol
and glucose, were correlated with attraction to food extract. During recovery from
stress, disassociation between physiology and behavior below particular thresholds of
65
physiological impact may reflect temporal variation in recovery based on the
importance ofthe particular behaviors to survival or performance.
Generally, all ofthe behavioral responses that were investigated in this study
recovered from stress by 5 hours, whereas all of the physiological measures still
indicated stress at this time. These results suggest that group association, feeding, and
predator avoidance capabilities were important behaviors for juvenile sablefish to
rapidly resume. The problem with using behaviors that recover quickly for !he
measurement of stress is that stress is cumulative, and the physiological system may
still be taxed by stress and less able to withstand an additional stress even though
behaviors appear normal (Schreck 2000).
As illustrated in this study, differences in the temporal dynamics of recovery
from stress, based on measures from various levels of biological organization, provide
insight into the physiological and behavioral capacities for recovery from stress, and
the relative hierarchical importance of particular behavioral or physiological
components to survival and performance. Another example of temporal variation in
behavioral and physiological recovery from stress is provided in a study by Pickering
et al. (1982) on the time course of recovery from acute handling stress in brown trout
(Salrna trutta): cortisol and lactate concentrations both peaked 2 hours after stress and
recovered to normal levels at 4 hours, glucose peaked at 4 hours and took 3 days to
return to normal, and stressed fish did not feed for 3 days after the stress. In a study
that compared feeding with physiology, adult sablefish towed in a simulated trawl for 4
hours did not recover normal feeding for 6 days, although cortisol had recovered by 3
days (Olla et al. 1997). These results differ from the current study with juvenile
sablefish, where feeding behavior recovered faster than physiological parameters. This
may be due to the greater severity of the stressor in the study on adult sablefish, or may
reflect the greater importance of feeding to faster-growing juveniles. In contrast to
studies where feeding behavior is slow to recover from stress compared with some
biochemical measures, predator avoidance behavior has been shown to recover faster
than the biochemical parameters measured. For example, predator avoidance
capabilities of coho salmon (0. kisutch) recovered from handling stress by 90 min
though cortisol levels were still high (Olla et al. 1992). Similarly, predator avoidance
66
in juvenile chinook salmon recovered by 1 hour after stress, even though cortisol,
glucose, and lactate did not recover until 6-24 hours (Mesa 1994). Gadomski et al.
(1994) also found that the stress of descaling juvenile chinook salmon did not impair
predator avoidance even though cortisol, glucose, and lactate concentrations in plasma
were elevated. The recovery of selected behaviors before physiological recovery
suggests the importance of these behaviors to survival or fitness. Behaviors that do not
recover until after physiological recovery has occurred may be those that are less
valuable to fitness than are the compromised components of the physiological system.
Differences in sensitivity to modifying factors also affect the correspondence of
indicators of stress. In the current study, feeding history and presence of a group of
conspecifics affected behavioral and physiological responses differently. For example,
feeding history generally altered the magnitude of the glucose and feeding responses,
but altered the rate of recovery of the lactate and cortisol responses rather than the
magnitude. In addition, group influence facilitated the recovery of activity but not the
visual food response. These patterns of disassociation provide insight into the
dynamics of the physiological responses and into the hierarchy of behavioral responses
in juvenile sablefish.
The importance of determining the effects of potential factors that can modify
assays for stress in order to control for these factors when assessing stress, or to
determine which factors may alleviate or aggravate stress was also illustrated in this
study. In general, recent feeding (1 day of food deprivation) was associated with either
lowered biochemical responses to stress or with faster recovery of biochemical values
towards normal levels when compared to responses of fish that had been deprived of
food for a longer period (5 days), suggesting that recent feeding alleviates the
physiological stress response in juvenile sablefish. Recent feeding was also associated
with depressed appetitive responses in control fish, thereby impairing the ability ofthe
assays of feeding behavior to detect differences related to stress. The capacity for the
stress response to be aggravated by social factors has been shown in rainbow trout (0.
mykiss), where subordinate status compromised physiological recovery from stress
(Pottinger and Pickering 1992). However, as juvenile sablefish exhibit strong social
attraction and are not aggressive, even during feeding (Sogard and Olla 2000), it was
67
hypothesized that the visual presence of feeding conspecifics would facilitate
behavioral and perhaps physiological recovery from stress. To the contrary, group
presence distracted isolates from feeding at times, and physiological measures tended
to be elevated over those of fish recovering alone. The attraction of isolates for the
groups was strongly evident, and physical separation may have been an additional
source of stress. Therefore, it would be instructive in the future to evaluate the
recovery of stressed fish within actual physical contact of unstressed conspecifics to
determine if this difference would promote facilitation of recovery. The physical
presence of conspecifics for a schooling species may also help to alleviate the stress of
perceived predation risk, which may be itself an additional stressor. For example,
when Atlantic salmon (s. safar) smolts were osmotically stressed, the presence of
predators worsened the physiological effect (Jarvi 1990).
Behavioral Responses
Typically, all of the behaviors assessed in this study were sensitive to stress yet
recovered quickly. In general, stress was associated with immediate depression in
general activity, appetitive behaviors, and swimming speed. These behaviors all
recovered to control levels by 5 hours. Feeding history and group influence treatments
altered the sensitivity of these assays
~y
affecting either baseline or stress-induced
responses, in concordance with behavioral strategies that would maximize fitness and
survival. Hunger motivation modified baseline levels of behaviors related to foraging
activity, with low hunger depressing responses in relation to the corresponding stressed
fish and high hunger increasing responses. The presence of groups during recovery of
stressed fish tended to facilitate the recovery of general activity and association with
group activities, which would be of theoretical benefit in terms of predator avoidance.
The hierarchical importance of group association over feeding was evident in the faster
recovery of group association in stressed fish, and in the overall competitive and
inhibitory effects that group presence had on the visual appetitive response.
Actions and decisions made during rec~)Very from stress are vitally important
for survival and minimizing the cost of stress on performance. Short-term survival is
68
of primary concern, followed by longer-term considerations such as growth and
reproduction. As a consequence, allocation of energy to behaviors involved with
predator avoidance theoretically is a priority during recovery from stress, followed by
behaviors such as feeding and social. interaction. The sensitivity of behaviors to stress
reflects their hierarchical level of importance to survival and performance. Fast growth
and schooling are traits ofjuvenile sablefish that heighten the importance of
resumption of normal feeding and social capacities. The importance of feeding and
social behaviors to juvenile sablefish was reflected in their behavioral responses to
stress, and in the roles that food deprivation and social isolation played in behavioral
recovery.
General Activity Patterns
The sensitivity of general activity to stress was illustrated by the obvious stressrelated lack of over-all activity during the 1 hour observation period, followed by
recovery to control levels by 5 hours. General activity patterns and variation were
useful in establishing behavioral baselines and developing appetitive assay protocols,
and provided valuable information about the dynamics of the feeding history and group
influence treatments. However, the individual variation and between-experiment
variation in activity that were found in this study cautions against the use of activity
levels as a precise measurement of stress.
Although swimming stamina has been used as a common indicator of stress (for
review see Beitinger and McCauley 1990), general locomotor activity has not been
typically used for this purpose. However, activity responses of fish to the stress of
being transferred to new environments are often considered when assessing acclimation
offish. For example, Henderson (1980) describes and reviews such behaviors as
freezing, hiding, and seeking the company of other fish as common responses during
adjustment periods. Hyperactivity may be initially noted in response to stressors such
as acid (Jones et al. 1987) and temperature changes (Olla et al. 1975) where fish may
be attempting to move away from the stressor, followed by hypoactivity when the
stressor cannot be avoided and physiological systems are impacted.
69
The depression of activity exhibited by stressed fish in this study is a typical
and adaptive response to acute stress. Depression of activity in response to stress
reduces predation risk and allows diversion of energy towards recovery. The
performance capacity of an animal is metabolically limited. The energy available for
performance is defined as the scope for activity, which is the difference between
maximum and minimum metabolic rates (Fry 1947). Environment or stress may
constrain performance capacity by increasing the load on the physiological system
(Schreck 1981). Long-term effects of stress place an allostatic load on physiological
systems that reflects the cost of adaptation (McEwen and Stellar 1993; McEwen 1998).
In a study by Mohamed (1982), handling stress resulted in lowered random activity and
elevated oxygen consumption in freshwater mullet (Rhinomugil corsula). Barton and
Schreck (1987) used oxygen consumption as a measure of metabolic cost incurred by
the response to acute stress in juvenile steelhead (S gairdneri), and found that a twominute stressful event caused at least a two-fold increase in metabolic rate. They
further calculated that even a minor stress could reduce the amount of energy available
for other activities by one-quarter. Davis and Schreck (1997) found that handling
stress was also metabolically costly for juvenile coho salmon, as measured by increases
in oxygen consumption, and that the metabolic cost increased with the severity of the
stressor, with exposure to air being the most costly of the stressors investigated.
Whether the lack of activity exhibited by stressed fish during the 1 hour
observation period was significantly different from controls or not was affected by
feeding history. In the first series of experiments, activity of control fish deprived of
food for 1 day was significantly lower than for control fish deprived of food for 5 days
during the 1,5, and 24 hour periods. Control fish deprived of food for 5 days also
spent a greater proportion of time in the upper sections of the experimental tanks than
did control fish deprived of food for 1 day. Since sablefish do not have swim bladders
and are negatively buoyant, time spent in the upper half of the tanks required constant
swimming, whereas time spent in the lower half of the tanks included resting time.
These results indicated a greater willingness of the fish motivated by greater hunger to
spend time on more energetic and risky behaviors, and affected the ability of general
activity to measure stress. The lack of detectable difference during the 1 hour
70
observation period between activity of the stressed and control fish deprived of food
for 1 day was likely due to the low activity of the controls as well as the stressed fish.
Food deprivation increases motivation to locate food sources. Increased activity levels
have the benefit of increasing the chance of finding food, but carry increased energetic
and risk of predation costs. Sogard and Olla (1996) found that as ration level declined
in walleye pollock (Theragra chalcogramma), motor activity increased, suggesting that
high activity was related to increased feeding motivation and searching behavior.
Similarly, rainbow trout on food-restricted diets were more active during feeding than
fish on satiation diets (Hojesjo et al. 1999).
The influence of group activity on the activity patterns of isolated fish was
clearly illustrated in this study. The initial lack of activity of stressed fish during the 1
hour observation period was not different from controls when the stressed fish were
recovering within view of groups in the first series of experiments. Here, the activity
of the stressed fish increased as the activity of the groups increased in response to
chemical and visual food stimuli. This increase was sufficient to make the activity of
the stressed fish recovering in view of groups similar to that of controls. Likely, the
stressed fish were responding to the increased activity of the groups rather than the
food stimuli, as this increase was not apparent in other treatments where fish were
recovering alone but in the presence of the stimuli. Activity levels did not recover to
control levels until at least 2 hours after the stress in fish recovering alone. The
increase in activity of the stressed fish following the increase of the groups' activity
also occurred in the second series of experiments, but was not quite as high as the
relatively high activity of the corresponding control fish during this period. The lack of
individual variation, the discrete increases in activity of fish within view of groups
closely mirroring the activity patterns of the groups over time, and the
disproportionately large amount of time that both control and stressed fish within view
of groups spent in sections of the tank closest to the groups clearly illustrated a strong
influence of group activity on the activity of the isolated fish, and the attraction of the
isolates for group association. In the case of the stressed fish, the motivation to
respond to the groups' activity recovered faster than appetitive behavior.
71
The benefits of social behavior include facilitation of feeding and predator
avoidance. Fish recovering from stress have increased energetic demands and are more
vulnerable to predators, making social benefits of even greater value. Isolated fish
within view of groups may use visual cues from group behavior to help make decisions
about their own feeding and predator avoidance behaviors. Isolated mullet (MugU
cephalus) in view of groups had high fish-to-fish attraction for groups of conspecifics,
and responded to visual cues provided by feeding or nonfeeding conspecifics to either
facilitate or inhibit their own feeding behavior (Olla and Samet 1974). Similarly,
isolated chum salmon (0. keta) in visual contact with groups ate most when groups
were also feeding, less when groups were not feeding, and least when groups were
alarmed (Ryer and OHa 1991). The desire of a schooling fish to be with a group may
increase with the perception of predator threat. For example, Sogard and alIa (1997)
found that walleye pollock responded to a visual predator threat by increasing group
cohesiveness. The perceived threat of predation may be heightened for fish recovering
from stress or in a novel environment. The comparative safety of being with a group
may help to offset the loss of capacity for predator avoidance in schooling types of fish
recovering from stress.
The large individual variation in the first experiment that was evident during
the 48 hour observation period of the treatments where fish were recovering alone
suggested a possible confounding factor that may have been related to the addition of
isolation stress. Isolation of individuals in schooling fish may be related to an increase
in fright behavior that is alleviated by the presence of conspecifics (Lemly and Smith
1985; Werner and Hall 1974). This amount of variation during the 48 hour observation
period was not apparent for individuals recovering within view of groups. Juvenile
sablefish are non-aggressive and schooling, and it is possible that isolation over time
increasingly influenced activity patterns.
For fish that had low activity levels prior to the introduction of chemical
extract, increases in activity were the predominant response, with most fish that
increased activity significantly also responding to the following visual food stimulus.
These results correspond with typical food-searching behaviors that are chemically
induced, where increases in swimming speed and turning aid in locating the food
72
source (Steven 1959; Pearson et al. 1980; L0kkeborg et al. 1995). However, for fish
that are already active, either a continuation or a reduction in swimming speed may be
required to accurately locate the food source. Moreover, due to the relatively small
size ofthe experimental tanks in c.omparison to fish size in this study, there was a
physical constraint on how fast fish could swim and tum. Fish that were highly active
before the chemical extract was introduced all responded by decreasing their activity.
This decrease in activity appeared related to an increase in searching behaviors. All of
these fish responded subsequently to the visual food stimulus, so it may be inferred that
they were appetitively responsive. Fish with medium levels of pre-extract activity also
responded to the visual food stimulus, but had only small increases or decreases in
activity in response to the chemical extract.
While individual variation in over-all activity and activity responses to food
stimuli was high for juvenile sablefish, such variation is inherent in behavioral research
and can be considered as potentially ecologically and evolutionarily important (for
review see Magurran 1993). Knowledge of individual variation provides insight into
potential plasticity and range in behavioral responses. For example, Gotceitas and
Colgan (1988) found significant individual variation in learning to forage on novel
food in juvenile bluegill sunfish (Lepomis macrochirus), and discuss the potential
importance of this variation in terms of environmental variation and evolution.
Variation in behavioral strategies in response to stress may be related to population
fitness. For example, rainbow trout that survived hypoxia had completely different
behavioral strategies for coping with the stress than non-survivors (Van Raaij et al.
1996).
Chemoreceptive and Visual Responses to Food
Both chemoreceptive and visual responses to food stimuli proved to be
sensitive indicators of stress. Depression of both of these appetitive responses in
stressed fish was evident at 1 hour, with recovery to control levels by 5 hours. This is
an adaptive response for an acute stress since it reroutes energy towards combating and
recovering from stress. It also may help to reduce the risk of predation, since a cost of
73
the movement and attention required by feeding is predation risk (Godin and Smith
1988; Lima and Dill 1990). However, if inanition continues it becomes maladaptive as
energy and vitamin reserves are depleted to a point where survival, growth,
reproduction and disease resistance are affected. Feeding motivation is a balance of the
costs of food acquisition and predation risks with the benefits of potential energy gain
as perceived by the animal (for reviews see Dill 1983; Lima and Dill 1990). Energy
from food is translated into performance capacity, and as optimal foraging theories
suggest, should be maximized (Hart 1993).
Chemoreceptive and visual responses to food were both sensitive to food
deprivation and group influence treatments. Food deprivation increases feeding
motivation. Social influence may facilitate feeding; thereby reducing energy needed
for foraging, and also may reduce predation risk. On the other hand, competition for
food may incite more risky behaviors to obtain food. As an example, foraging in
bluntnose minnows (Pimephales notatus) increased with shoal size, decreased with
predator presence and increased with hunger (Morgan 1988). Stress adds conflict to
the balance by increasing vulnerability to predation as well as increasing energy
demands. A stressful experience increases metabolic rate and oxygen consumption
(Barton and Schreck 1987). Illustrating the consequences of increased energetic
demands, the energetic stress of parasitization caused sticklebacks (Gasterosteus
aculeatus) to flee shorter distances for shorter periods of time from a predator threat,
and to resume feeding sooner than nonparasitized fish (Godin and Sproul 1988).
In general, chemoreceptive and visual responses to food stimuli were well
correlated in this study. Fish that responded to the chemical extract usually responded
to the visual stimulus as well. This was especially true for the food deprivation
treatments, but less so for the group influence treatments. The strong influence ofthe
groups' activity on behavior of the individual fish resulted in disassociation between
the chemoreceptive and visual response assays. More fish within view of groups had
responses to the chemical extract than subsequently ate. In part, this may have been
due to the false chemoreceptive responses in stressed fish elicited by group behavior,
and while the chemoreceptive responses of the groups appeared to facilitate
corresponding responses in isolates, the attention of the isolates was often so distracted
74
by the feeding of the group that they did not notice the food on their own side of the
tanks. In this case, the social transfer of information may have indicated to the isolate
that the food source was in the vicinity of the group.
This study established chemoreceptive response to food extract as a sensitive
and useful indicator of stress in sablefish. Chemoreception assays of stress in fish have
typically been used to evaluate avoidance/attractance and sublethal effects of water
pollutants (Jones et al. 1987; Jones and Hara 1988; Birge et al. 1993; Kasumyan and
Morsi 1998). Studies incorporating chemoreceptive response as a measure of physical
stress are limited.
Levels of food deprivation affected the ability of the chemoreceptive assay to
indicate stress due to effects on the behavior of control fish. During the 1 hour
observation period, only a small percentage « 20%) of stressed fish deprived of food
for 1 or 5 days responded to food odor. These responses were likely false responses
based on normal activity increases, as they were similar to the small percentage of false
responses found in the treatment where no odor was introduced. The small percentage
of stressed fish responding to extract was less than the high percentage (80-100%) of
control fish responding for fish deprived of food for 5 days. However, stressed fish
were not different from controls for fish deprived of food for 1 day due to the low
number of control fish responding (about 40%). So, while there was a stress-related
depression of chemoreceptive response, the difference from controls was not
significant when appetitive motivation was also reduced in controls.
The group influence treatments also affected the ability of the chemoreceptive
assay to measure stress, but in this case due to the effects on the behavior of the
stressed fish. During the 1 hour observation period, none of the stressed fish
recovering alone responded to the chemical extract, whereas over 40% of the stressed
fish recovering within view of conspecifics responded. Since less than 20% of these
fish ate during the subsequent visual assay, it is likely that these were mostly false
responses instigated by the abrupt and strong increases in activity exhibited by the
groups in response to the chemical stimulus. As a result, the apparently high
percentage of stressed fish responding when in view of groups was not significantly
different from controls. In this case, the strong motivation of the isolated stressed fish
75
to respond to group activities, which was already evident at 1 hour of recovery, was
confounded as an appetitive response.
While chemoreception is an integral part"of the arousal, search, and ingestion
phases of feeding behavior, its role in stimulating searching behavior was chosen as the
best method for evaluating appetitive response in this study. Searching for food
requires active energy expenditure, and is therefore an informative measure of
motivational status, as well as capability of responding to food stimuli. In a study by
L0kkeborg et al. (1995), searching behavior in sablefish was strongly elicited in
response to squid extract, with the response threshold being affected by feeding
history. A drawback of the use of searching activity as a measure of chemoreceptive
response is that the activity patterns defined as being search related may be
misconstrued as being related to food stimulus when there may be alternative
explanations, such as the influence of conspecific behavior.
The visual response to soaked squid incorporated arousal, searching, and
ingestion phases of feeding, as the visual response was ultimately measured by
consumption of the food. Stress may impact any or all of the phases, making food
consumption a potentially very sensitive measure of stress. Handling stress has been
shown to cause reduction or cessation of feeding in fish (Pickering et al. 1982; Mesa
and Schreck 1989; Olla et al. 1997). Fish that were actively searching were more
likely to see the food compared with fish that were resting on the bottom of the tanks,
or had their attention focused elsewhere. Consequently, the visual response was also
sensitive to factors affecting activity patterns.
As with the chemoreceptive response, levels of food deprivation affected the
ability of the visual response to indicate stress in the first series of experiments due to
differences in responses of control fish. Only a small percentage (20%) of stressed fish
deprived of food for both 1 and 5 days responded to the soaked squid during the 1 hour
observation period, indicating a depressed motivation to feed. This was significantly
less than the number of control fish deprived of food for 5 days that responded (>
80%), but did not differ from the control fish deprived of food for 1 day, as only about
40% of these fish responded. In contrast, both the control fish deprived of food for 1
and 5 days in the second series of experiments had relatively high numbers of fish
76
responding (> 80%) during the 1 hour period. This
hi~h
percentage of visual response
may be related to the greater activity, and therefore greater chance of seeing food,
observed in these fish compared with the sinne treatment in the first series of
experiments.
While group presence seemed to facilitate the return of activity levels of
stressed fish in association with group activity, and therefore of apparent
chemoreceptive responses, group presence did not facilitate the recovery of food
consumption. Therefore, unlike the chemoreceptive response, the presence of
conspecifics did not affect the ability of the visual assay to indicate stress by
influencing the responses of the stressed fish. During the 1 hour observation both
stressed fish that were alone and those with groups exhibited less than 20% visual
response. These results indicate that the presence of the groups did not facilitate
recovery of the visual response to food. In fact, there appeared to be a decline in visual
response for both control and stressed fish recovering within view of groups from 5 to
48 hours in the first series of experiments, and from· 1 to 5 hours for controls in the
second series. While some instances were noted where the group's interest in the food
falling on the individual's side seemed to incite increased interest by the individual,
there were also several instances where the individual fish were so focused on the
group's activity that the food was never noticed. Individual fish were almost
constantly oriented towards the groups. It is possible that separation from the group
was distractive and even stressful, and became more so over time, resulting in
responses that were contradictory to what was expected.
Juvenile sablefish are gregarious and non-aggressive, but very competitive
when food is present. Therefore, it was thought that the social stimuli provided by a
feeding group of conspecifics would facilitate the recovery of feeding in stressed
individuals. Facilitation of feeding has been documented as one of the reasons for
gregarious behavior. Benefits of feeding with a group include decreased predation risk
conferred by group vigilance and enhancement of food resources through information
transfer among individuals. Illustrative of these· benefits, growth rates were greater for
juvenile chum salmon in visual contact with conspecifics than for isolates (Davis and
alIa 1992). Similarly, individual walleye pollock juveniles exploited a greater number
77
of food patches when in groups of6 than when isolated (Baird and Olla 1991).
Juvenile walleye pollock in groups also began feeding sooner than isolates, and
individuals in groups used social cues to gather information about food location (Ryer
and Olla 1992). Alternatively, the presence of competing conspecifics may incite
feeding at the expense of taking greater risks. For example, competitor presence has
been correlated with decreased sensitivity of feeding behavior in the presence of
predation risk for juvenile coho salmon (Dill and Fraser 1984).
Swimming Speed
The swimming capacity of stressed fish attempting to escape was generally
impaired at 1 hour of recovery, but had recovered to control levels by 5 hours.
Theoretically, predator avoidance would also have been impaired, suggesting relevance
for short-term survival. For fish compromised by stress, the return of adequate
predator escape capabilities is of paramount importance. However, this recovery may
be limited physiologically by the reallocation of energy to cope with stress, and by
injuries incurred during the stress. Predator avoidance has been shown to be impaired
by handling stress (Olla et al. 1992; Mesa 1994; Olla et al. 1995; Olla et al. 1997). As
an indirect measure of predator avoidance, juvenile chinook salmon took longer to
reach cover when stressed (Sigismondi and Weber 1988). In the present study, the
capture nets served as "predators", and swimming speed away from the nets was an
indirect measure of capacity for predator avoidance. Impairment of predator avoidance
further increases the need for any advantage that group membership may confer, and
may have been a motivating factor in the quick recovery of social interaction that
occurred in stressed fish within view of groups. Recent feeding also impaired
swimming speed. During the 1 hour observation period, the speed of control fish that
were fed the day before was sufficiently slowed as to not differ from that of stressed
fish.
78
Physiological Responses
Plasma cortisol, glucose, and lactate were sensitive indicators of acute stress,
with elevations occurring by the 1 hour sampling time, and recovery by 24 hours after
the stress. However, these factors were affected by feeding history, and suggested'
effects of group influence as well. Days of food deprivation affected peak responses
and recovery times of these measures, with 1 4ay of food deprivation being related to
lower response values or quicker recoveries compared with 5 days of food deprivation.
Response values for solitary controls had recovered to baseline values by the 5 hour
observation period, whereas values for all of the other group influence treatments were
still elevated.
Cortisol, glucose and lactate responses differed in their sensitivities to stress,
food deprivation, and group influence. As a result, there was a non-correspondence
among these assays. The mechanisms of disassociation were alterations of peak
responses and recovery rates. Due to the role of cortisol in maintaining glucose levels,
these two biochemical indices are more likely to correspond with each other than with
lactate. Cortisol and glucose were more sensitive to the less severe stress of transfer
than lactate. Cortisol exhibited the most variation between replicates. Glucose was the
most sensitive to food deprivation levels, followed by cortisol, then by lactate. Cortisol
and glucose elevations indicated influences of group presence on stress levels in
relation to baseline values, but lactate did not. These differences in sensitivities to
stress and modifYing factors reiterate the need for considering more than one assay for
assessing stress, especially if variables that may affect these measures exist or are
unknown.
Cortisol
Basal and peak concentrations of plasma cortisol in juvenile sablefish were
similar to values found for other teleosts. Basal values for fish sampled directly from
holding tanks were less than 25 ng/ml, peak stress values at the 1 hour sampling time
were greater than 100 ng/ml, and values for fish that had recovered to levels no longer
79
significantly different from basal concentrations were less than 75 ng/ml. These values
are similar to those found in stressed and unstressed adult sablefish (Olla et al. 1998).
Common cortisol levels in uns~ressed, non-reproductively active teleost fish are less
than 30-40 ng/ml, and peaks are 40-200 ng/ml or more in stressed fish (for reviews see
Wedemeyer et al. 1990; Barton and Iwama 1991).
Cortisol elevations of stressed fish were highest at the 1 hour sampling time and
had recovered to control values within 24 hours, which is a typical time course for
recovery. The primary role of cortisol in responding to acute stress is the immediate
initiation of processes involved with intermediary metabolism and energy mobilization,
and with maintaining homeostasis (for reviews see Van Der Boon et al. 1991;
Wendelaar Bonga 1997; Mommsen et al. 1999). Likely due to this role, cortisol in
plasma often peaks quickly (within minutes) in response to an acute stress, followed by
a relatively fast recovery to basal levels (within hours), even though secondary and
tertiary responses may still be apparent for days or even weeks (Molinero et al. 1997;
and for reviews see Schreck 1981; Schreck et al. 1997).
Cortisol was very sensitive to stress, registering moderate increases to 100-150
ng/ml in response to just the stress of transfer which was experienced by the control
fish. These levels were no longer significantly elevated above baseline levels at 5
hours for most of the treatments. Cortisol, in this case, reflected the severity as well as
the presence of stress. Cortisol is often very sensitive to stress, reflecting even minimal
stress such as removal of cohorts from a holding tank (Laidley and Leatherland 1988;
Young and Cech 1993a; Molinero et al. 1997), or transfer from one tank to another
(Strange and Schreck 1978). As another example, serum cortisol in plaice
(Pleuronectes platessa) was significantly affected by whether tanks were uncovered or
covered before blood sampling, although there was no difference in serum glucose and
the other serum components measured (White and Fletcher 1986).
While peak cortisol levels in response to stress were not affected by level of
food deprivation in this study, recovery of cortisol from stressed levels was faster for
fish fed the day before in comparison with fish fed 5 days before. At the 5 hour
sampling time stress-induced cortisol levels increased with relation to the level of food
deprivation. Since basal cortisol values of fish sampled directly from holding tanks did
80
not reflect effects of food deprivation level, these trends may be related to a
physiological mechanism, or psychological perception of stress, which may help to
alleviate the stress response.
Cortisol levels, both basal and stress-induced, have been shown to be
influenced by nutrition and food deprivation, however, with contradictory conclusions.
Henrique et al. (1998) suggested that the enhancements in cortisol response to stress
that were associated with Vitamin C supplementation in seabream (Sparus aurata)
were indicative of ascorbate playing a role in corticosteroid metabolism. White
suckers (Catostomus commersoni) deprived of food for 4 weeks had a more rapid
cortisol response to acute stress, and a slower return to baseline values than fed fish
(Bandeen and Leatherland 1997). On the other hand, Barton et al. (1988) found lower
resting levels of cortisol in juvenile chinook salmon deprived of food for 20 days than
in fed fish, and noted a trend towards cortisol responses to stress being lower in food
deprived fish. These conflicting results may reflect the different treatments, food
deprivation times, and species. Dietary content and duration of food deprivation have
different metabolic consequences for different species at different life history periods.
Interestingly, a direct link between cortisol and feeding has been established: chronic
elevation in plasma cortisol in juvenile rainbow trout is associated with negative effects
on feeding behavior, growth rate, condition, and efficiency of food conversion, and
with greater variability in feeding patterns (Gregory and Wood 1999).
As with the behavioral responses, it was expected that the recovery of
physiological responses to stress would be facilitated by the presence of an unstressed
feeding group. However, similar to the behavioral responses, there was some
indication that recovering within view of groups could have been an added stressor,
perhaps due to the physical separation. The only treatment with cortisol levels low
enough to not be significantly different from basal values was the solitary control
treatment, suggesting that even the control fish in view of groups were somewhat
stressed at 5 hours.
While there is substantial evidence that agonistic behavior (Ejike and Schreck
1980; Pottinger and Pickering 1992; Wilson and Roys 1994; Fox et al. 1997) and
crowding stress (Wedemeyer 1976; Pickering and Stewart 1984; Leatherland and Cho
81
1985) are sufficiently stressful to raise cortisol levels in fish (for review see Schreck
1981), whether social facilitation of physiological measures occurs in fish is unknown.
For species with agonistic encounters, isolation may be less stressful than being a
subordinate. However, in species that are schooling and where social facilitation of
behaviors occurs, it is possible that being with conspecifics could help to lessen the
impact of a stressful event or allow quicker recovery, and that isolation may aggravate
effects of stress.
Glucose
Plasma glucose levels in juvenile sablefish in this study were sensitive to stress
and fell within typical ranges for stressed and unstressed teleosts. Basal glucose values
of fish sampled from holding tanks were around 40 mg/dl, peak values for stressed fish
at the 1 hour sampling time were around 150 mg/dl, and values for fish that had
recovered sufficiently to be no longer different from controls were around 60 mg/dl.
These values were similar to the basal and peak values of adult sablefish that ranged
from about 40-180 mg/dl in a study by Olla et al. (1998). The common range of basal
glucose levels is around 40-80 mg/dl, and for peak stress levels is 120-250 mg/dl (for a
review of typical teleost blood glucose values see Chavin and Young 1970).
The highest glucose levels were found in stressed fish at the 1 hour sampling
time with recovery to control levels occurring by 24 hours. Elevations of plasma
glucose in teleosts are usually rapid, though slightly slower (minutes to hours) and
often oflonger duration (hours to days) than plasma cortisol elevations (Barton et al.
1986; Robertson et al. 1987; Thomas and Robertson 1991; and Mesa 1994). Although
differences between the timing of cortisol and glucose responses were not measured in
this study, the time course for recovery of glucose levels following stress fell within
common ranges for teleosts.
The hyperglycemic response for both control and stressed fish fed the day
before was depressed compared with fish fed 5 days before. While glucose was
moderately elevated for control fish deprived of food for 5 days in response to the
transfer stress alone, therefore measuring both the presence and severity of stress, this
82
capability was not expressed in fish fed the day before. At the 5 hour sampling time
there was a significant trend for stressed-induced glucose levels to increase with
relation to the level of food deprivation. Basal levels of glucose obtained from fish
taken directly from holding tanks did not vary with food deprivation levels, indicating
that the effect of food deprivation on glucose was not manifested in unstressed fish.
The depressed hyperglycemic response in recently fed fish may have been linked to the
corresponding decreased severity of the cortisol response, as cortisol plays a role in
maintaining glucose levels during stress.
Basal and stress-induced levels of plasma glucose are affected by feeding
history. For example, short-term (1 week) food deprivation in coho salmon results in
hyperglycemia as liver glycogen stores are mobilized, followed by normal glycemic
levels as liver glycogenolysis ceases after longer periods of food deprivation (3 weeks)
(Sheridan and Mommsen 1991). The dynamics of food deprivation effects on glucose
can therefore affect the capacity of the hyperglycemic response to stress. Long periods
of food deprivation may deplete liver glycogen stores and therefore reduce the capacity
for glycogenolysis to release glucose for energy. Plasma glucose in unstressed rainbow
trout was higher in fed fish than in those deprived of food for 6 weeks, and food
deprived fish indicated a slower and depressed glucose response after stress than fed
fish (Farbridge and Leatherland 1992). Striped bass (Marone saxatilis) deprived of
food for up to 2 months were still able to produce a hyperglycemic response to stress,
though of reduced magnitude compared with fed fish (Ridley et al. 1997). Barton et al.
(1988) also found a reduction in magnitude of the hyperglycemic response to stress
when chinook salmon were deprived of food for 20 days compared with fish that had
been fed, and for fish on lower lipid diets compared with fish on higher lipid diets.
These studies all incorporate relatively long periods (greater than 1 week) of food
deprivation, which presumably reduces the capacity for a hyperglycemic response to
stress as a result of reduced glycogen stores available for energy mobilization. Less is
known about the effects of shorter-term (less than 1 week) periods of food deprivation
on the glucose stress response. However, the results of the current study indicated a
decreased hyperglycemic response in recently fed fish compared with those deprived of
83
food for several days, an opposite trend to what has been found for longer periods of
food deprivation.
As with cortisol, glucose indicated that recovering within view of groups was a
potentially added stressor. Glucose levels of solitary control fish did not differ from
basal values. However, glucose levels of all of the other treatments were still elevated
at the 5 hour sampling.
Lactate
Plasma lactate was sensitive to stress in juvenile sablefish. Basal lactate values
for fish sampled from holding tanks were less than 10 mg/dl. Peak lactate values at the
1 hour sampling time were around 200 mg/dl. These basal and peak values were
within the ranges found for unstressed and stressed adult sablefish (Olla et al. 1998).
However, the peak values were higher than those commonly found for teleosts.
Concentrations of plasma lactate typically range from less than 10 mg/dl to around 100
mg/dl in teleosts (Beamish 1966, 1968; Gadomski et al. 1994; Molinero et al. 1997).
Lactate levels of stressed fish were still elevated (80-150 mg/dl) at 5 hours but
recovered to basal levels by 24 hours. Like glucose, plasma lactate levels often peak
within minutes to hours of the onset of stress, and return to pre-stress levels within
hours to days (Beamish 1966, 1968; Young and Cech 1993b).
Unlike cortisol and glucose, lactate was not sensitive to the transfer stress that
control fish received, likely due to the lack of any prolonged struggle associated with
the transfer process. Lactate is increased by stress responses that require exhaustive
activity such as fast swimming, struggling, or hypoxia. Therefore, lactate may not be
as sensitive to stress, compared with cortisol and glucose, for stressors that are not
severe enough to induce anaerobic metabolism. Alternatively, plasma lactate may have
been rapidly cleared to baseline levels by the 1 hour sampling time.
Recent feeding was associated with faster recovery of lactate after stress.
While peak lactate levels at the 1 hour sampling time were similar for both stressed fish
deprived of food for 1 and 5 days, lactate levels of stressed fish deprived of food for 1
day had recovered to lower levels at the 5 hour sampling time than for fish deprived of
84
food for 5 days. At the 5 hour sampling time there was a significant trend for stressedinduced lactate levels to increase with relation to the 1, 3, and 5 day food deprivation
levels. The mechanism for faster clearing of lactate from plasma in recently fed fish is
unknown, but likely reflects physiology less compromised by stress than for fish that
had been deprived of food. As with glucose, lactate response to stress can be affected
by energy reserves. For example, Atlantic cod (Gadus morhua) fed 72 hours before
exercise had lower lactate responses than fish fed 12 hours earlier (Beamish 1968).
Similarly, unfed striped bass had lower lactate responses to angling stress than did fed
fish (Ridley et al.1997). These studies suggest that the capacity for a lactate response,
and possibly recovery rate as well, can be reduced with food deprivation time. As an
indirect example, food deprivation (3 days) did not affect lactic acid or glucose
responses to stress in striped bass, but recovery of glycogen stores was slower than for
fed fish (Reubush and Heath 1996).
Concluding Remarks
This work points out the effects that nutritional state and social environment
may have on behavioral and physiological responses to stress. Caution is urged to
researchers that there is a need to be cognizant of potential modifying effects of
nutrition and social environment, both when comparing results of different studies, and
when choosing appropriate measures for assessing stress.
85
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99
APPENDIX
100
13.0
.-
(a)
0
.,U 12.0
---=
'-'
~
~
11.0
~
0
'Y
'Y
61
\l
10.0
-.,
~
•
c. 10.5
~
0
1t
11.5
~
e
•
Experimental Series 1
12.5
•
0
'Y
\l
•
•
\l
0
•
\l
\l
9.5
•
•• •
'Y
~
'Y
~
I Day
5 Days
Solitary
Social
No Food Stimulus
9.0
Sep
13.0
Oct
Nov
Dec
Jan
Feb
Mar
Apr
May
Apr
May
(b)
Experimental Series 2
.- 12.5
U
.,'-'
---=
~
eo::
c.
12.0
11.5
•
~
e
11.0
0
~
~
'Y
\l
•
10.5
10.0
Sep
600
-
400
eJJ
300
'-'
...-=
Oct
Nov
Dec
Jan
Mar
Feb
(c)
500
eJJ
,-
•
1 Day
5 Days
Solitary
Social
Holding Tank
•
0
0
Experimental Series 1
Experimental Series 2
(5)0
eO
i •
• 0
~~
0
~
~ 200
100
0
200
250
300
350
400
450
Total Length (mm)
Figure 1. Mean temperature values recorded during experimental series 1 (a) and 2 (b),
and relationship between weight and total length for experimental series 1 and 2 (c).
Date:
Treatment:
Fish Intro: (= time zero)
Pre-food stimulus
Activity Time/Block
Time
1
2
0-1
Fish:
Tank:
3
Stressed/Control:
Recovery time:
Extract Intro: (time 0 intro + 30 seconds)
Post extract
Activity Time/Block
Time
1
3
2
0-1
Sablefish Behavior: Experimental Series 1
=
4
4
Squid Intro:
Time eaten:
Post squid
Activity Time/Block
Time
1
2
3
1-2
1-2
1-2
2-3
2-3
2-3
3-4
3-4
3-4
4-5
5-6
4-5
5-6
4-5
5-6
6-7
6-7
7-8
6-7
7-8
7-8
4
0-1
8-9
8-9
8-9
9-10
9-10
9-10
12-13
12-13
15-16
15-16
12-13
15-16
18-19
21-22
18-19
18-19
21-22
21-22
24-25
24-25
24-25
27-28
27-28
27-28
30-31
30-31
33-34
33-34
36-37
35-36
39-40
36-37
37-38
38-39
39-40
40-41
41-42
42-43
43-44
44-45
45-46
Figure 2. Sample data sheet for recording of activity patterns for experimental series 1.
--o--
Date:
Treatment:
Fish Intro: (= time zero)
P re-f 00 d sf Imu Ius
Activity Time/Block
Time
1
0-1
Fish:
Tank:
2
3
4
Stressed/Control:
Recovery time:
Extract Intro: (time 0 =intro + 30 seconds)
P os t ext ract
Activity Time/Block
Time
1
2
3
Sablefish Behavior: Experimental Series 2
4
0-1
Squid Intro:
Time eaten:
Pos t SCUI°d
Activity Time/Block
3
Time
1
2
4
0-1
1-2
1-2
1-2
2-3
2-3
2-3
3-4
:3-4
3-4
4-5
4-5
4-5
5-6
5-6
5-6
6-7
6-7
6-7
7-8
7-8
7-8
8-9
8-9
8-9
9-10
9-10
9-10
35-36
36-37
37-38
38-39
39-40
40-41
41-42
42-43
43-44
44-45
Figure 3. Sample data sheet for recording of activity patterns for experimental series 2.
..o
tv
Number of replicates for all treatments performed for experimental series 1 (a) and experimental series 2 (b). The two
numbers within a block shown for experimental series 2 refer to the number of replicates used for activity patterns (left), and
the number of replicates used for behavioral (chemoreceptive and visual appetitive responses, and swimming speeds) and
physiological (cortisol, glucose and lactate) assays for stress (right).
EXPERIMENTAL SERIES 1 (BEHAVIOR): REPLICATES
EXPERIMENT
Food Deprivation
TREATMENT
1 Day
5 Day
Group Influence
Solitary
Social
No Food Stimulus CONTROL/STRESS
Control
Stress
Control
Stress
Control
Stress
Control
Stress
Control
Stress
OBSERVATION PERIOD (HOURS)
1
48
5
24
7
5
7
7
7
7
5
7
7
6
7
7
7
7
6
7
9
9
5
9
9
9
9
5
8
7
7
7
8
8
7
7
8
8
8
8
EXPERIMENTAL SERIES 2 (BEHAVIOR AND PHYSIOLOGY): REPLICATES
OBSERVATION PERIOD (HOURS)
EXPERIMENT
TREATMENT CONTROL/STRESS
1
5
24
Food Deprivation 1 Day
15/6
9/6
3/2
Control
Stress
15/6
9/6
3/3
5 Day
Control
16/6
10/6
4/4
Stress
16/6
10/6
4/4
Group Influence
Solitary
Control
6/6/6
-/Stress
6/6/6
-/Social
Control 7/7/7
-/7/7
Stress
7/-
-/-
.......
oVol