AN ABSTRACT OF THE THESIS OF
Sarah C. Lupes for the degree of Master of Science in Fisheries Science presented on
May 25, 2004.
Title: Capture-related Stressors Impair Sablefish, Anoplopoma fimbria, Immune System
Function
Abstract approved:
Redacted for Privacy
Carl B. Schreck
Sablefish, Anoplopomafimbria, is a valuable North Pacific Ocean species caught
in several commercial fisheries and is often discarded due to size or catch limits.
Managers must account for the mortality of discarded fish to assess fish populations and
harvest impacts, yet discard mortality rates of specific fisheries are generally unknown.
Delayed mortality is one source of undetected mortality in discarded fish. The
observance of delayed mortality in discards during laboratory and field studies suggest
that capture stressors may induce physiological modifications that result indirectly in
future mortality from stress andlor disease. Since the imposition of a stressor disrupts a
fish's capability to maintain immunological function, understanding the capacity of the
immune system to function after an acute bycatch stressor may be important in assessing
the survival of discarded fish and further understanding the mechanisms behind delayed
mortality.
The main objective of this study was to describe the immunological health of
sablefish exposed to capture stressors. First, experiments were performed to obtain the
conditions necessary for [3H]-thymidine incorporation into lymphocytes sensitive to B
and T cell mitogens. Then, in laboratory experiments designed to simulate the capture
process, sablefish were subjected to various stressors that may influence survival: towing
in a net, hooking, ecologically relevant temperatures of seawater and air, and air exposure
time. Following the imposition of stress, the immunological assay was used to assess
immune function.
The results demonstrated that regardless of fishing gear type, exposure to elevated
seawater temperatures or air times, [3H]-thymidine incorporation into leukocytes from
stressed sablefish was significantly diminished in response to the T cell mitogen
concanavalin A or the B cell mitogen Iipopolysaccharide. Seawater or air temperatures
were not found to significantly influence immune responses. The duration and severity
of the capture stressors applied in our study were sufficient to induce significantly
elevated levels of cortisol and glucose. However, there was no difference in the
magnitude of plasma cortisol and glucose responses among stress treatments. These data
suggest that immunological suppression occurs in sablefish subjected to capture-related
stressors. Functional impairment of the immune system after exposure to capture-related
stress is a potential reason for delayed mortality in discarded sablefish. As fishes are
returned to the ocean, their ability to perform at the whole organism level may be
diminished for an extended period of time and the cumulative strain from maintaining
physiological adaptation can reduce their ability to sustain normal immunological
functions. Consequently, immunological impairment may predispose a discarded fish to
disease and eventual death. However, further studies are needed to determine if delayed
mortality in discarded sablefish can be caused by increased susceptibility to infectious
agents resulting from stress-mediated immunosuppression.
© Copyright by Sarah C. Lupes
May 25, 2004
All Rights Reserved
CAPTURE-RELATED STRESSORS IMPAIR SABLEFISH, ANOPLOPOMA FIMBRIA,
IMMUNE SYSTEM FUNCTION
by
Sarah C. Lupes
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented May 25, 2004
Commencement June 2005
Master of Science thesis of Sarah C. Lupes presented on May 25, 2004.
APPROVED:
Redacted for Privacy
Maj or Professor, representing Fisheries Science
Redacted for Privacy
Head of the Department of Fisheries and Wildlife
Redacted for Privacy
Dean of the Giaduate School
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
Sarah C. Lupes, Author
ACKNOWLEDGEMENTS
To all my committee members and mentors for their support, expertise, and
guidance. I sincerely thank: my major advisor Carl Schreck, for his overall vision in this
project, and his dedication to the academic growth of his students which is shown
through his continuous involvement and copious advice throughout this project, and
Michael Davis, for his generous wealth of knowledge, and his implementation of all
laboratory experiments. Their guidance was invaluable to me; Bori 011a for his
continuous insights and enthusiasm that carried this work to completion; Scott Heppell
for his perspective of commercial fisheries and Chris Bayne for his insights in fish
immunology and data interpretation; and Mary Arkoosh for her critical advice and
interpretations in the development of the immune assay.
I am grateful to everyone in the Oregon Cooperative Fish and Wildlife Research
Unit for their generosity in providing support for this research. I am indebted to Beth
Siddens for her unlimited support, and for her patience in teaching me how to run assays.
I also thank Carisska Anthony for her technical and emotional support, her consistent
energy in sampling, and her help in "fishing" out samples from the HMSC Nitrogen
storage tank. Amarisa Marie and Ichiro Misumi were both instrumental in providing
assistance throughout this endeavor. Thanks to Grant Feist, Shaun Clements, David
Jepsen, and Mark Karnowski for their willingness to take a moment when I had general
lab or computer questions.
I am also very appreciative of Paul Reno and Prudy Caswell-Reno for their
hospitality in providing me with the laboratory space for sampling and completion of the
immune assay. They were both willing to work around my sampling schedules. Ethan
Clemons also dedicated his time to the development of the immunological assay and
taught me pertinent laboratory techniques. In addition, the microbiological department at
OSU, especially Peter Bottomley, was very generous in allowing me the use of their
equipment.
Thanks to all my old and dearest of friends, as well as those who I have made at
OSU for your encouragement and support throughout this endeavor. A very special
thanks to Jennifer Hebding and Nicolas Romero for keeping me sane. With deep
appreciation, I thank my parents Tom and Marsha Lupes and my brothers Matthew and
Daniel, who have always cheered me on through all my adventures. I am so thankful to
have such a loving family. Thank you Dexter, Missy and Zoe!
This project was made possible by the generous support of Oregon Sea Grant.
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, Oregon in collaboration with the Department
of Fisheries and Wildlife, Oregon Cooperative Fish and Wildlife Research Unit at
Oregon State University, Corvallis, Oregon.
CONTRIBUTION OF AUTHORS
Carl Schreck contributed in developing the experimental design, data analysis and
interpretation, and editing of the manuscripts. Michael Davis contributed in developing
the experimental design, providing expertise in laboratory capture-related stressors,
subjecting sablefish to stressors, and editing of the manuscripts. Bori 011a assisted with
the experimental design and provided expertise in working with sablefish. Mary Arkoosh
assisted in the development of the immune assay, interpretation and analysis of the
immunological data, and editing of the first manuscript.
TABLE OF CONTENTS
Page
INTRODUCTION
.1
Sablefish ................................................................................................ 2
Capture-related Stressors ................................................................. 3
The Stress Response .......................................................................... 4
AN IN VITRO CULTURE SYSTEM FOR SABLEFISH, ANOPLOPOMA
FIMBRIA,
LYMPHOCYTE MITOGEN-INDUCED PROLIFERATION ................. 9
Abstract ..................................................................................... 9
Introduction ................................................................................... 9
Material and Results ....................................................................... 12
Collection and maintenance of sablefish ...................................... 12
Isolation and cultivation of pronephric leukocytes ........................... 12
Osmolality measurements ....................................................... 14
Reagents and mitogens ......................................................... 14
Kinetics and mitogenic dose assays ........................................... 15
Leukocytedensity ............................................................... 16
Temperature ...................................................................... 19
Hypotonic lysis verses density gradient centrifugation ..................... 19
Variability of mitogenic responses ............................................ 21
TABLE OF CONTENTS (Continued)
Page
Discussion
.21
References ................................................................................. 27
CAPTURE-RELATED STRESSORS IMPAIR SABLEFISH, ANOPLOPOMA
FIMBRIA, IMMUNE SYSTEM FUNCTION ................................................ 31
Abstract .................................................................................... 31
Introduction ................................................................................ 32
Materials and Methods ................................................................... 35
Fish collection and maintenance ............................................... 35
Experimental procedure ......................................................... 35
Dataanalysis ..................................................................... 38
Results ..................................................................................... 39
Discussion ................................................................................. 46
References ................................................................................. 56
CONCLUSION .................................................................................... 61
REFERENCES .................................................................................... 62
LIST OF FIGURES
Figure
Proliferative responses of sablefish pronephric leukocytes
to various concentrations of lipopolysaccaride (LPS) or
concanavalin A (Con A) ................................................................. 17
2.
Stimulation indices (SI) for various numbers of sablefish
pronephric leukocytes activated with 25 tg/ml of either
lipopolysaccaride (LPS) or concanavalin A (Con A) ................................ 18
Effects of in vitro culture temperatures on the proliferative
responses of sablefish pronephric leukocytes to 25 tg/ml of
either lipopolysaccharide (LPS) or concanavalin A (Con A) ....................... 20
4.
Proliferation responses of sablefish pronephric leukocytes to
25 tg/m1 of either lipopolysaccaride (LPS) or concanavalin
A (Con A) subjected to density gradient centrifugation or
hypotoniclysis ........................................................................... 22
Mean (+SEM) stimulation indices (SI) of sablefish subjected
to 2 h of hooking or towing at 8°C , followed by 15 mm air
exposure at 10 or 16 °C in comparison to controls (unstressed
at8°C) ..................................................................................... 40
6.
Mean (+5EM) stimulation indices (SI) of sablefish towed for
2 h at 8 °C followed by 15 mm exposure to an elevated seawater
temperature of 10 or 16 °C in comparison to controls (untowed
at8°C) .....................................................................................
7.
42
Mean (+SEM) stimulation indices (SI) of sablefish subjected
to different air exposure times (15 or 30 mm) and air
temperatures (10 or 16 °C) ............................................................... 43
Mean (+SEM) plasma cortisol concentrations (nglml) of
sablefish subjected to various stressor treatments in comparison
to controls (unstressed at 8 °C) ........................................................ 44
9.
Mean (+SEM) plasma glucose levels (mg/dl) of sablefish
subjected to various stressor treatments in comparison to
controls (unstressed at 8 °C) ............................................................ 47
LIST OF TABLES
esponses of leukocytes from pronephros of 25
tive 2 year-old sablefish to 25 tg/ml of either
Laride (LPS) or concanavalin A (Con A) on day
re ............................................................................ 23
CAPTURE-RELATED STRESSORS IMPAIR SABLEFISH, ANOPLOPOMA
FIMBRIA, IMMUNE SYSTEM FUNCTION
INTRODUCTION
The unaccounted mortality associated with the capture and return of non-targeted
fish to the ocean, as discarded bycatch, is one of the primary issues currently affecting
commercial fisheries management. Although some important steps have been taken to
abate bycatch mortality, such as modifications in fishing gear technology (Broadhurst
2000), discard mortality rates of specific fisheries are generally unknown and often
speculative (Chopin et al. 1995; Pascoe 1997; NMFS 2003). Delayed mortality is one
source of undetected mortality in discarded fishes. The observance of delayed mortality
in discards during field and laboratory studies indicates that capture stressors may induce
physiological modifications that result indirectly in future mortality from predation,
stress, and/or disease (Davis 2002). Since the imposition of a stressor disrupts a fish's
capability to maintain immunological functions (for reviews see: Barton and Iwama
1991; Wendelaar Bonga 1997; Schreck 2000), understanding the capacity of the immune
system to function after exposure to a bycatch stressor may be important in assessing the
survival of discarded fish and further understanding the mechanisms behind delayed
mortality.
In order to assess the effects of capture-related stress on the immune function of
sablefish, Anoplopomafimbria, an important commercial species, we needed to initially
establish an assay to characterize or monitor the immunological health of sablefish. In
the first the first chapter of this thesis, we report a number of experiments that evaluated
2
the conditions necessary to optimize a tritiated-thymidine lymphocyte incorporation
assay sensitive to B and T cell mitogens. In the second chapter, our objective was to
assess the immunological health of sablefish exposed to capture stressors. Therefore, in
laboratory experiments designed to simulate the capture process, we subjected sablefish
to various stressors that may influence survival: towing in a net, hooking, ecologically
relevant temperatures of seawater and air, and air exposure time. After the application of
stressors, the proliferation assay described in the first chapter was used to assess the
sablefish immune system function.
Sablefish
Sablefish is an important North Pacific Ocean species that when not targeted in a
commercial fishery (McFarlane and Beamish 1983a) is often discarded due to size or
catch limits. Sablefish are susceptible to overexploitation due to their long lives with
ages over 50 years regularly recorded (Kimura et al. 1983; McFarlane and Beamish
1 983b) and their highly migratory nature (Maloney and Heifetz 1997; Rutecki and Varosi
1997) with distribution from northern Mexico to the Gulf of Alaska, westward to the
Aleutian Islands, and into the Bering Sea (Wolotira et al. 1993). Sablefish abundance is
dependent on strong year classes (McFarlane and Beamish 1992) with juveniles
providing the primary source of recruitment into the sablefish fishery (Sigler et al. 2001).
During near-shore residency, juvenile survival is associated with sea temperature (Sogard
and 011a 1998) and food abundance (McFarland and Beamish 1992). Juvenile sablefish
are very susceptible to trawl fisheries during near-shore residency and their incidental
3
discard can adversely effect sablefish recruitment (Sampson et al. 1997; Sigler et al.
2001). The size-selective price structure of the commercial industry and the minimum
size limits also result in the discard of smaller fish potentially leading to greater discard
mortality (Schirripa and Methot 2002; Davis and Parker 2004).
Capture-related Stressors
During the capture process, mortality in fish may result from the interactions of
various stressors including initial capture, environmental factors, and handling (Davis
2002). Temperature is one of the primary interacting factors that can contribute to
increased discard mortality rates. Sablefish are often caught at depths ranging from 155
to 1350 m with long-line or trawl capture methods (011a et al. 1998; Schrippa and Methot
2002). During the retrieval of fishing gear, sablefish are often exposed to rapid
temperature changes ranging from 6 to 17 °C (011a et al. 1998). Fishing in warmer
months will result in sablefish exposed to elevated surface seawater temperatures that
may induce greater rates of bycatch mortality. Laboratory and field studies have
documented increased stress and mortality related to increased temperature (Barton and
Iwama 1991; 011a et al. 1998; Davis et al. 2001; Davis 2002, 2004 in review).
Air exposure is also a critical factor that can increase the level of stress and
mortality during the bycatch process (Davis 2002). Air exposure is equivalent to the
amount of time a fish remains on deck and the temperature of air. The handling time of a
fish is dependent on the size of the catch and the type of gear employed, which can lead
to air exposures ranging from several minutes to an hour. The air temperature to which a
fish is exposed on deck will vary in accordance with the time of year fished. Fishing in
an el niño year and in warmer seasons will result in exposure to warmer temperatures of
air on deck. While sablefish are sensitive to increased temperature, they are considered
relatively resilient in air, as mortality in laboratory experiments did not occur until after
30 minutes of air exposure (Davis and Parker 2004).
An important biological factor that modulates stress and mortality is the size of
the fish. As commercial value is related to size in the sablefish fishery, there is an
incentive to retain larger fish and discard smaller, less profitable fish. In the laboratory,
mortality resulting from bycatch stressors differed between size classes of sablefish with
smaller fish being more sensitive to the capture process (for review see: Davis and Paker
2004). Smaller sablefish reached higher body core temperatures more quickly than larger
fish (Davis 2001). Consequently, smaller sablefish reached 100% mortality at a lower
temperature when exposed to gradients of increasing temperatures in laboratory
experiments (Davis and Parker 2004).
The Stress Response
The stress response results in physiological and behavioral changes that can be
quantitatively measured to reflect the degree of stress a fish experiences (Barton and
Iwama 1999). Responses to stress have been classified according to primary, secondary,
and tertiary changes that reflect the different levels of biological organization (Mazeaud
and Mazeaud 1981; Wedemeyer and McLeay 1981; Wedemeyer et al. 1990). Primary
responses are neuroendocrine changes dominated by the hypothalmic-sympathetic-
chromaffin cell axis and the hypothalmic-pituitary interrenal axis (Colombo et al. 1990;
Wendelaar Bonga 1997).
After a stressful stimulus is perceived by the central nervous
system (CNS), catecholamines (CA), epinephrine and norepinephrine, are secreted from
chromaffin cells in the pronephros (anterior kidney), stimulating immediate responses in
the cardiovascular system, respiratory and osmoregulatory functions, and metabolism
(Wedelaar Bonga 1997). Within the HPI axis, the hypothalamus controls the release of
the corticotrophin-releasing hormone (CRH) and the thyrotropin-releasing hormone
(TRH). These hormones stimulate the pituitary to secrete adrenocorticotropic hormone,
alpha melanocyte-stimulating hormone (u-MSH) and -endorphine, which then controls
the production of cortisol in the interrenal cells of the head kidney. Cortisol is important
in maintaining homeostasis and mobilizing energy reserves. The elevation of
corticosteriods, mainly cortisol, is a widely used indicator of monitoring the severity and
duration of the primary stress response. Measurement techniques for cortisol are well
established, and are functionally significant in physiological processes affecting fish
health (Donaldson 1981; Barton and Iwama 1991).
The secondary stress responses are the blood and tissue modifications that result
from the actions of the primary response (specifically CA and cortisol). Secondary
changes are apparent in blood and tissue chemistry, hydrominera! balance, and
hematology, and can include elevated blood sugar and lactate levels (Wedemeyer et al.
1990). Hyperglycemia is initially induced by CA stimulation of glycogenolysis in the
liver, and may then be maintained by cortisol action. Measures of glucose are also quick
and easy to perform and have been frequently used as indicators of secondary stress
responses (Barton et al. 1986).
Tertiary or whole animal (Barton and Iwama 1991) stress responses include
changes in metabolic rate, health, behavior, growth, reproduction success, and mortality
rates (Pickering 1981; Colombo et al. 1990; Wedemeyer 1990). They have the ability to
predict the effects of stress at the population or community levels (Wedemeyer et al.
1984). Though the tertiary changes may be more difficult to assess, they have the highest
"ecological relevance" (Adams 1990). Comparisons of measures of stress responses
from different biological levels will help to enhance the ecological value of a particular
measure and will further illustrate the dynamics of the stress response.
Situations that push physiological systems beyond homeostasis can result in
what Sterling and Eyer (1998) have defined as allostasis: the ability of an organism to
increase or decrease vital functions to achieve a new form of stasis (Sterling and Eyer
1988; McEwen 1998). Yet, the cumulative strain on the organism from maintaining this
allostatic response to stress can result in an allostatic load (McEwen and Stellar 1993),
and consequently reduce the organism's ability to maintain normal physiological
functions (Pruett 2003).
The interactions of various capture stressors such as trawling, hooking,
temperature and air exposure have been studied in laboratory sablefish with the stress
response measured as changes in behavior, physiology, and mortality (011a et al. 1997,
1998; Davis et al. 2001; Spencer 2000; Davis 2002, Davis and Parker 2004). Gradients
of increased bycatch stressors amplified physiological indices of stress such as plasma
cortisol, glucose, lactate, potassium and sodium (011a et al. 1997, 1998; Davis 2002).
Though the magnitude of the stress response was related to the intensity of the stressor
7
there was no correspondence between mortality and physiological measures of stress
(Davis et al. 2001).
Since the imposition of a stressor disrupts a fish's capability to maintain
immunological function, understanding the capacity of the immune system to function
after a bycatch stressor may be important in assessing the survival of discarded fish and
further understand the mechanisms behind delayed mortality It is known that stress can
have long-lasting negative effects on the general health of mammals (Dhabhar and
McEwen 2001) and teleosts (for reviews see: Anderson 1990; Barton and Iwama 1991,
Schreck 1996; Wendelaar Bonga 1997; Weyts et al. 1999). Correlations of
immunosuppression and disease outbreaks in fish have been demonstrated in various
laboratory settings (Anderson 1990).
Several techniques have been developed and used for monitoring the immune
competence of fish (Anderson 1990). Immunological assays have been standardized for
many freshwater (Rosenberg-Wiser and Avtalion 1982; Clem et al. 1984; Kaattari and
Yui 1987; DeKoning and Kaatarri 1991) and marine fish (Reitan and Thuvander 1991;
Arkoosh et al. 1994; LoPresto et al. 1995) to demonstrate changes due to natural and
anthropogenic stressors. Various cellular immune functions in fish have been
investigated to determine the status of the immune system (Ellasesser and Clem 1986;
Yin et al. 1995; Ortuno et al. 2002). The proliferative responses of leukocytes to a
mitogen can be considered a reasonable approximation to show that the immune system
of a fish is capable of functioning (Ellasesser and Clem 1986; Luft et al. 1991; Faisal and
Hargis 1992). However, to date, there are no established techniques to characterize or
monitor the immunological health of sablefish. An assay to measure the responses of fish
leukocytes to in vitro stimulation is one method of assessing the function of the sablefish
cellular immune system. The ability to assess and measure the immune responses of fish
in laboratory-simulated bycatch experiments may lead to further insights regarding the
delayed mortality of bycatch (Davis 2002). Further comprehension of the effects of the
interactions of capture and environmental stressors on sablefish are critical for providing
understanding of what controls delayed discard mortality
9
AN IN VITRO CULTURE SYSTEM FOR SABLEFISH, ANOPLOPOMA FIMBRIA,
LELTKOCYTE MITOGEN-INDUCED PROLIFERATION
Abstract
An in vitro cell culture system that supports the proliferative responses ofpronephric
leukocytes to the B cell mitogen lipopolysaccharide (LPS) and the T cell mitogen
concanavalin A (Con A) was established for sablefish, Anoplopomafinibria, a marine
teleost. Proliferation was assessed by leukocyte [3H]-thymidine incorporation and
expressed as a stimulation indices calculated as the ratios of counts per minute (cpm) of
mitogen-stimulated cultures divided by the cpm of unstimulated cultures. Minimal
Essential Media made isotonic to sablefish plasma and supplemented with fetal calf
serum aided mitogen stimulation. Pronephric leukocytes exhibited similar responses to
both mitogens, with maximum stimulation achieved after three days of culture at 17 °C.
A higher degree of proliferation occurred in response to LPS when leukocytes were
purified from erythrocytes using density gradient centrifugation versus hypotonic lysis.
In contrast, the magnitude and kinetic responses of leukocytes to Con A was unaffected
by hypotonic lysis, suggesting that sablefish leukocytes that are activated by LPS could
be more sensitive to changes in the isotonicity of the surrounding media. These data
imply that the in vitro mitogenic stimulation of pronephric leukocytes can be utilized for
monitoring the immunological health of sablefish in aquaculture and natural populations.
Introduction
Sablefish, Anoplopoma JImbria, is an important North Pacific Ocean species in
commercial fisheries and aquaculture. Intensive aquaculture practices that can lead to
stress-mediated immunosuppression and the need to develop procedures to control
disease necessitate the development of methods to measure the immune functions of
sablefish. Sablefish are also caught in several commercial fisheries and are often
discarded due to size or catch limits. This species is susceptible to overexploitation due
to their long lives, with ages over 50 years regularly recorded (Kimura et al. 1983;
McFarlane and Beamish 1983) and their highly migratory nature (Maloney and Heifetz
1997; Rutecki and Varosi 1997) with distribution from northern Mexico to the Gulf of
Alaska, westward to the Aleutian Islands, and into the Bering Sea (Wolotira et al. 1993).
As there is substantial discarding of sablefish in the Northeastern Pacific commercial
fishery industry (Sampson et al. 1997), the ability to assess and measure the immune
responses in laboratory-simulated bycatch experiments may lead to further insights
regarding discard delayed mortality (Davis 2002). However, currently there are no
validated techniques to characterize or monitor the immunological health of sablefish.
Sablefish are members of the large order Scorpaeniformes that share the common
character of a suborbital stay, formed by the second infraorbital crossing the cheek from
the orbit to the preopercle (Bond 1996). Anoplopomatidae have been largely unstudied
physiologically, with no information currently on disease resistance capacity.
Investigations of the immune functions in species not commonly studied could facilitate
further insights into the teleost immune system.
Several techniques have been developed and used for monitoring the immune
competence of fish (Weyts et al. 1999). The in vitro stimulation of fish leukocytes from
11
the peripheral blood, thymus, pronephros, and spleen is one of the most commonly used
methods to assess the function of the cellular immune system (Luft et al. 1991; Faisal and
Hargis 1992). The proliferative responses of leukocytes can vary with fish species,
mitogen type and environmental factors, as shown in channel catfish, Ictalurus punctatus
(Clem et al. 1984; Sizemore et al. 1984; Lufi et al. 1991), English sole, Pleuronectes
retulus (Arkoosh et al. 1994), rainbow trout, Oncorhynchus mykiss (Etlinger et al. 1976;
Tillit et al.1988; DeKoning and Kaatarri 1991; Hardie et al. 1993), carp, Cyprinus carpio
(Rosenberg-Wiser and Avta!ion 1982; Hamers 1994; Troutaud et al. 1995), sea bass,
Diecentrarchus labrax L. (Jeney et al. 1994; Galeotti et al. 1999), red drum, Sciaenops
ocellatus (LoPresto et al. 1995), Chinook salmon, Oncorhynchus tshawytscha (Kaattari
and Yui 1987; DeKoning and Kaattari 1992), and Atlantic salmon, Salmo salar (Reitan
and Thuvander 1991). As conditions for the maintenance and stimulation of leukocytes
vary for each species of fish, modification of previous immunological techniques are
required to study the immunological health of a different species (Wolf 1979; Galeotti et
al. 1999).
The proliferative responses of leukocytes to a mitogen can be considered a
reasonable first approximation to show that the immune system of a fish is capable of
functioning (Ellasesser and Clem 1986). However, to date, there are no established
techniques to characterize the immunological health of sablefish. The basic aim of this
research is to establish an in vitro culture system that supports the mitogenic response of
sablefish pronephric leukocytes. We report a number of experiments that evaluated the
conditions necessary to maximize a tritated-thymidine lymphocyte incorporation assay
12
sensitive to B and T cell mitogens. Studies of sablefish cellular immune function can
then be used to monitor the health of sablefish in aquaculture and in natural populations.
Methods and Results
Collection and maintenance of sablejlsh
Juvenile sablefish {20-40 mm total length (TL)] were collected in spring of 2001
offshore of Newport, Oregon and reared at Hatfield Marine Science Center in circular
tanks (2.0 m diameter, 0.8 m depth, 31401 volume) supplied with sand-filtered and TJV
sterilized flow-through seawater (30-32°/ salinity, 10-13°C, 02 >90% saturation) at a
replacement rate of 101. min'. Fish were fed to satiation on pelletized salmon food
three times a week. During the second year, the fish were maintained at 3 0-40 fish in
large circular tanks (4.5m diameter, 1.0 m depth) supplied with seawater (201. min
1,
30-32°/ salinity, 02 >90% saturation, 5.5-7.5°C) and fed a diet of squid, Loligo
opalescens, until satiated two times a week.
Isolation and cultivation ofpronephric leukocytes
Non-reproductive 2-year-old sablefish were rapidly netted and immediately
placed in a lethal dose (400 ppm) of tricaine methanesulfonate (MS-222). The caudal
peduncle of each fish was severed and the blood drained in an effort to reduce red blood
cells count in the tissues. Individual pronephros were harvested into tissue culture media
13
(TCM) under aseptic technique. Cells were separated from the surrounding tissue by
gently teasing the tissue with the end of a 3 ml syringe plunger through a 40 Jtm Falcon
cell strainer. The single cell suspension was diluted to a total volume of 10 ml with
TCM in 50 ml conical tubes (Becton Dickinson) and then washed by centrifugation at
500g for 15 mm at 17 °C. Subsequently, the supernatant was decanted, the cellular pellet
resuspended with 1 Omi of TCM and centrifuged again at 500g for 15 mm at 17 °C. Using
a hemocytometer, leukocytes from the single cell suspension were counted using the
trypan blue exclusion method, and adjusted to 2 x i0 cells/mi.
Erythrocytes and leukocytes from the kidney were separated according to
techniques described by Kaattari and Holland (1990) and adapted by Arkoosh et al.
(1994). Briefly, 10 ml of a whole kidney cell suspension (2 x
i07
cells/mi) were slowly
layered over an equivalent volume of Histopaque in 50 ml conical tubes and centrifuged
at 500 g for 45 mm at 17 °C. The buffy layer of leukocytes at the interface was removed
with a pasteur pipette, TCM added for a total volume of 10 ml, and the cells centrifuged
at 500g for 15 mm at 17 °C. The leukocyte pellet was resuspended in 10 ml of TCM and
centrifuged again. After the second wash, the supernatant was aspirated and the pellet
resuspended with 0.5 ml TCM. Using the trypan blue exclusion method to determine
viability, the isolated populations of leukocytes were counted with a hemocytometer and
diluted to 5 x 1
7
cells/ml with TCM. For the proliferation assay, cells were added in
triplicate to flat bottom 96 well tissue culture plates (Becton Dickinson) at 5 x 1 0
cells/mi in 100 pJ of TCM, and stimulated with 100 p.! aliquots of the appropriate
concentration of mitogen. The cultures were maintained at 17 °C in a humidified
incubation chamber (C.B.S. Scientific Co.) containing a blood gas mixture (10% 02, 10%
14
CO2, and 80% N2) and fed on alternative days with 10
tl
of a modified nutritional
supplement (Kaattari et al. 1986).
Osmolality measurements
Osmolalities of freshly separated plasma from 15 individual sablefish and the
prepared TCM were measured using a vapor pressure osmometer.
Reagents and mitogens
The medium for isolation and maintenance of leukocytes was prepared as
described in Mi!ston et al. (2003) and Misumi (2004) but adjusted according to sablefish
blood osmolality of approximately 380 mosm. The TCM consisted of a 7% heatinactivated fetal bovine serum (FBS), 1% L-glutamine (bOX), and 9% tumor cocktail in
Minimum Essential Media (MEM). The tumor cocktail was prepared by adding 7.5 g
dextrose, 75 ml essential amino acids (50X), 100 ml non-essential amino acids (bOX),
and 100 ml sodium pyruvate (bOX), to 630 ml of MEM. The mixture was adjusted to a
pH of 7.0 with 10 N NaOH before adding 8.5 g of sodium bicarbonate, 340 mg penicillin,
0.2 mg m1
1
streptomycin sulfate, 0.1 mg mt gentamycin, and 34 jil 1321
mercaptoethanol. The cocktail was dispensed through a 0.45 j.tm filter, aliquoted into
45m1 per conical tube and stored at 20 °C. MEM was purchased from Invitrogen Co.,
and all other reagents were purchased from Sigma Chemical Co.
15
The nutritional supplement was prepared by a modification of the method of
Kaattari and Holland (1990). Fifteen microliters of TCM was added to 7.5 ml of heatinactivated fetal bovine serum, 0.347 ml HEPES buffer, 1.0 ml guanosine in TCM
(1 .0mg/mi), and a 1 ml mixture containing equal parts adenosine, uridine, and cytidine in
TCM (1.0 mg/mi). The solution was adjusted to a pH of 7.4 with 10 N NaOH, filtered
through 45 im filter (Corning), and stored at 20 °C.
Mitogens were purchased from Sigma Chem. Co. Concanavalin A (Con A) a
polyvalent carbohydrate-binding protein from Canavalia ensiformis and
lipopolysaccharide (LPS) from the bacterium Echerichia coli serotype 0 55 :B5 were
obtained as aseptic powders and reconstituted with TCM to a stock solution (10 mg/mi).
The stock solution was diluted further to obtain the appropriate concentrations of mitogen
per well, and filtered (0.45 p.m filter) before use.
Kinetic and mitogen dose response assays
To determine the optimal dose of mitogen and to establish the peak day of
mitogen-induced proliferation, pronephric leukocytes from three sablefish were incubated
in triplicate at 17 °C with concentrations of 5, 25, 150, and 250 jig/well of LPS or 0.5, 5,
25, 50 jig/well of Con A. On days 0,2,4, or 6 of culture, 1 jiCurie (jiCu) of[3H]thymidine in 10 ul of nutrient supplement was added to each well. Twenty-four hours
after the [3HJ-thymidine was added, the leukocytes were harvested from their wells with a
Wallac Tomtec cell harvester onto a glass fiber filtermat (Wallac). The filtermat was
dried and sealed in a plastic filter bag with 4.5 ml of optiphase scintillation fluid
16
(Waliac). Proliferation was assessed by [3H]-thymidine incorporation measured as
counts per minute (cpm) by a Wallac liquid scintillation counter. Data were derived
from triplicate determinations for each fish. The proliferative responses, over
background, induced by either LPS or Con A were expressed as stimulation indices (SI),
calculated as the ratio of mean counts per minute (cpm) of mitogen-stimulated cultures
divided by the mean cpm of non-stimulated cultures. Maximum mitogenic responses for
both LPS and Con A were observed on day three of culture (Figure 1). A concentration
of 25 jig/mi for each mitogen was determined as the dose required to create a functioning
assay.
Leukocyte density
To determine the leukocyte density that produced the greatest SI, pronephric
leukocytes ranging from concentrations of 5 x 1 O to 5 x 1
7
cells/mi were stimulated in
triplicate with the optimal concentration of either LPS or Con A and incubated at 17 °C.
The cells were subsequently harvested on the culture day that produced the greatest
mitogenic response. One jiCu of {3H]-thymidine was added to the leukocytes activated
with either LPS or Con A on day 2 of incubation and the cells were harvested and
counted on day 3 as described above. A density of 5 x 106 leukocytes/mi produced the
greatest proliferative response to LPS and Con A (Figure 2) and was used in subsequent
studies.
17
10
8
6
4
2
a)
0
E
10
Con A (jig/well):
8
6
4
2
0
0
1
2
3
4
5
6
7
Incubation period (days)
Figure 1: Proliferative responses of sablefish pronephric leukocytes to various
concentrations of lipopolysaccaride (LPS) or concanavalin A (Con A). The proliferative
responses are expressed as stimulation indices calculated as the ratios of [3HJ-thymidine
uptake of the mitogen stimulated cultures divided by the [3H]-thymidine uptake of the
non-stimulated cultures. Data are derived from the means of three sablefish (± SEM)
with triplicate determinations for each fish. Control cultures (without mitogen) had a
mean count per minute of less than 400.
V
0
I
5x107
5x106
5x105
5x104
Leukocytes/mL
Figure 2: Stimulation indices (SI) for various numbers of sablefish pronephric leukocytes
activated with 25 g!m1 of either lipopolysaccaride (LPS) or concanavalin A (Con A).
Leukocytes were incubated at 17 °C and harvested on day three of culture. The
proliferative responses are expressed as SI calculated as the ratios of {3H]-thymidine
uptake of the mitogen stimulated cultures divided by the [3H]-thymidine uptake of the
non-stimulated cultures. Data are derived from the means of three sablefish (+SEM) with
triplicate determinations for each fish. Control cultures (without mitogen) had a mean
count per minute of less than 400.
19
Temperature
The leukocyte density, mitogen concentration, and duration of culture yielding the
maximum mitogenic proliferation were used to determine if a different in vitro
temperature would enhance the mitogen-induced proliferative response. The leukocytes
were cultured at three temperatures 8 °C, 12 °C, and 17 °C. Cells were pulsed with 1xCu
[3H]-thymidine on day two of culture, harvested twenty-four hours later and counted.
Sablefish kidney leukocytes assessed on day 3 of culture maintained a maximum SI for
both LPS and Con A at 17 °C (Figure 3). At 8 °C and 12 °C the mitogenic responses to
LPS were approximately at a SI of 1.0, while at 12 °C a SI of greater than 2.0 was
maintained for Con A (Figure 3).
Hypotonic lysis versus density gradient centrifugation
To determine if a greater SI could be produced with hypotonic lysis instead of
density centrifugation, leukocytes were separated from erythrocytes as described by
Crippen et al. (2001). Briefly, 2 ml of whole cell suspension was diluted with 9 ml sterile
distilled water, the erythrocytes lysed for 20 s, and the cells returned to isotonicity with 1
ml lox phosphate-buffered saline (PBS). The suspension was centrifuged at 500 g for 7
mm at 17 °C, the supernatant aspirated and the pellet resuspended in 2 ml of TCM.
Isolated leukocytes at a density of 5 x 106 were stimulated with 25 tg/ml of LPS or Con
A and kinetic assays were performed in triplicate at 17 °C. On days 0, 2, 4, or 6 of
incubation, the cultures were pulsed with 1 p.Cu [3H]-thymidine in 10 p.1 of nutrient
20
4
C
E
c/D2
8
12
17
Incubation temperature (°C)
Figure 3: Effects of in vitro culture temperatures on the proliferative responses of
sablefish pronephric leukocytes to 25 tg/m1 of either lipopolysaccharide (LPS) or
concanavalin A (Con A). Leukocytes were incubated at 8 °C, 12 °C, and 17 °C and
harvested on day three of culture. The proliferative responses are expressed as
stimulation indices calculated as the ratios of [3H]-thymidine uptake of the mitogen
stimulated cultures divided by the [3H]-thymidine uptake of the non-stimulated cultures.
Data are derived from the means of three sablefish (+ SEM) with triplicate
determinations for each fish. Control cultures (without mitogen) had a mean count per
minute of less than 400.
21
supplement, harvested 24 h later, and counted. The greatest SI for LPS was induced on
day 3 of culture with cells separated by density gradient centrifugation (Figure 4). While
the LPS proliferative response was suppressed following hypotonic lysis, the magnitude
and the kinetics of the response to Con A was relatively unaffected by hypotonic lysis
(Figure 4).
Variability of in vitro mitogenic responses
Using the culture conditions previously determined to be optimal, the degree of
variability in the proliferative response to a mitogen was assessed in 25 non-reproductive
2-3 year-old sablefish. Pronephric leukocytes of sablefish were stimulated in triplicate
with either 25 tg!ml of LPS or Con A, pulsed with [3H]-thymidine on day 2 of culture
and harvested 24 hours later. The stimulation indices ranged from 1.7 to 7.8 for LPS and
1.9 to 10.9 for Con A, with a mean SI of 5.0 and 5.8 for LPS and Con A, respectively
(Table 1.1).
Discussion
Leukocytes from the pronephros of the marine teleost Anoplopomafimbria can be
stimulated to proliferate in vitro with mitogens that are designated as B or T cell
activators in mammals (Ellis 1977). In this study, in vitro cell culture conditions were
defined with the mitogens LPS and Con A that will allow for future evaluation of the
22
Incubation period (days)
Figure 4: Proliferation responses of sablefish pronephric leukocytes to 25 tg!m1 of either
lipopolysaccaride (LPS) or concanavalin A (Con A) subjected to density gradient
centrifugation or hypotonic lysis. The proliferative responses are expressed as
stimulation indices calculated as the ratios of [3H]-thymidine uptake of the mitogen
stimulated cultures divided by the [3H]-thymidine uptake of the non-stimulated cultures.
Data are derived from the means of three sablefish (± SEM) with triplicate
determinations for each fish. Control cultures (without mitogen) had a mean count per
minute of less than 400.
23
Table 1.1: Proliferative responses of leukocytes from pronephros of 25 non-reproductive
2 year old sablefish to 25 ig/ml of either lipopolysaccharide (LPS) or concanavalin A
(Con A) on day three of culture. The proliferative response for each individual fish is
expressed as a stimulation index calculated as the ratio of [3H]-thymidine uptake of the
mitogen stimulated cultures divided by the [3H}-thymidine uptake of the non-stimulated
cultures. All non-stimulated cultures (without mitogen) had a mean cpm less than 500.
Stimulation index with mitogen
Fish
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Mean (SE)
LPS
ConA
6.2
4.0
3.6
5.6
4.2
1.9
5.5
7.4
1.9
5.6
6.8
4.8
4.4
3.4
3.4
4.0
2.1
6.4
1.7
6.3
5.9
5.4
9.9
6.2
10.9
5.5
8.2
4.8
3.9
5.5
4.2
9.8
8.4
7.0
1.8
5.1 (1.9)
5.8 (2.3)
5.1
3.9
4.9
7.8
5.1
3.7
5.1
3.1
7.0
6.7
8.3
24
immunological health of sablefish. The sablefish culture system employed the process
of adapting mammalian cell culture procedures and medium, which is very similar to the
methods used by other teleost systems developed for assessing leukocyte function.
Modifications to the TCM included the addition of sodium chloride to be isosmotic with
sablefish plasma and the supplementation of 7% heat-inactivated FCS. Although some
studies have revealed that the utilization of homologous plasma in leukocyte cultures
enhanced the mitogenic response in salmonid pronephros (DeKoning and Kaatari 1992),
and the peripheral blood leukocytes of channel catfish (Faulmaim et al. 1983), other
studies showed that leukocytes from marine species can proliferate in media
supplemented with only FCS (Faisal and Hargis 1992; Arkoosh et al. 1994; Galeotti et al.
1999).
Both Con A and LPS at a concentration of 25 jig/mi, induced similar increases in
mitotic activities in sablefish pronephric leukocytes when compared to their respective
controls. However, in the pronephros of different species the mitogenic stimulation may
vary with the type of mitogen presented. Leukocytes from the pronephros showed a
differential response to mitogens in chinook salmon (Leith et al. 1984) and Atlantic
menhaden, Brevoortia tyrannus (Faisal and Hargis 1992), with cells exposed to LPS
responding better than those receiving T cell mitogens, Con A or phytohemagglutinin
(PHA). Con A failed to induce any significant mitotic stimulation in pronephric
leukocytes in sea bass (Galeotti et al. 1999). Although the magnitudes of the responses
of sablefish pronephric leukocytes to Con A and LPS are lower than SI reported for
salmonids (Kaattari and Yui 1987; DeKoning and Kaattari 1992), they are similar to
those reported for English sole (Arkoosh et al. 1994), plaice, Pleuronectes platessa
(Grimm 1985), and Atlantic menhaden (Faisal and Hargis 1992). These interspecific
variations could be due to differences in mitogen-receptors on leukocytes or differences
in leukocyte populations within the pronephros of different species. The responsiveness
of the pronephric leukocytes in sablefish may also be reflective of the cultural conditions.
As previously demonstrated in carp (Le Morvan-Rocher et al. 1995) and bluegill,
Lepomis macrochirus (Cuchens and Clem 1987), the mitogenic response in sablefish was
affected by in vitro temperature. In channel catfish (reviewed in Bly and Clem 1991), the
magnitude of proliferative response of peripheral blood leukocytes to LPS was relatively
independent of in vitro temperature while the responses to Con A were suppressed at
lower temperatures (Clem et al. 1984). In our studies, sablefish proliferative responses to
Con A were clearly inhibited at both 8 and 12°C, while pronephric leukocytes were
minimally stimulated at 12 °C by LPS. However, whether this temperature sensitivity is
due to changes in mitogen kinetics or magnitude cannot be addressed with our data. Bly
and Clem (1991) emphasized the importance of in
vivo
temperature in the
in
vitro
activation of fish leukocytes, channel catfish cells responded to Con A with maximum
stimulation at an in vitro temperature 5 °C greater than the
in vivo
holding temperatures.
Sablefish acclimated at approximately 8 °C produced the greatest stimulation to both LPS
and Con A at an in vitro temperature 9 °C greater than their
in vivo
temperature.
Typically, teleost leukocytes from tissues are isolated by a density gradient
centrifugation through a medium such as Percoll or Histopaque. Crippen et al. (2001)
described a technique that uses hyptonic lysis to separate leukocytes from peripheral
blood and pronephros of salmonids. The present study demonstrates that sablefish
leukocytes isolated by hypotonic lysis were unable to respond to LPS. Separation
26
techniques employing hypotonic lysis should be exercised with caution when measuring
the proliferative ability of leukocytes to LPS, especially in marine teleosts.
Although
the relative magnitude and kinetic response to Con A was relatively unaffected by
hypotonic lysis, sablefish leukocytes that are activated by LPS could be more sensitive to
changes in the isotonicity of the surrounding media.
The present study demonstrates that pronephric tissue is suitable for use in
in vitro
assays to evaluate the proliferative responses of sablefish leukocytes to classic
mammalian B and T cell mitogens. The development of an
in vitro
culture system for
sablefish leukocytes will allow for future assessment of the function of sablefish cellular
immune system in aquaculture and natural populations.
27
P efrn re
Arkoosh, M. R., E. Clemons, and E. Casillas. 1994. Proliferative response of English
sole splenic leukocytes to mitogens. Transactions of the American Fisheries
Society 123:230-241.
Bly, J. E., and L. W. Clem. 1991. Temperature-mediated processes in teleost immunity:
in vitro immunosuppression induced by in vivo low temperature in channel
catfish. Veterinary Immunology and Immunopathology 28:365-377.
Bond, C. 1996. Biology of Fishes, 3' edition. Orlando, Florida.
Clem, L. W., E. Faulmann, N. W. Miller, C. F. Ellsaesser, and C. J. Lobb. 1984.
Temperature-mediated processes in teleost immunity: differential effects of in
vitro and in vivo temperatures on mitogenic responses of channel catfish
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Crippen, T. L., L. M. Bootland, J. C. Leong, M. S. Fitzpatrick, C. B. Schreck, and A. T.
Vella. 2001. Analysis of salmonid leukocytes purified by hypotonic lysis of
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Cuchens, M. A., and L. W. Clem. 1987. Phylogeny of lymphocyte heterogeneity II:
differential effects of temperature of fish T-like and B-like cells. Cellular
Immunology 34:219-230.
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lymphocytes requires homologous plasma for optimal responsiveness. In Vitro
Cellular and Developmental Biology 27A: 381-386.
DeKoning, J., and S. L. Kaattari. 1992. Use of homologous salmonid plasma for
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Syndrome. Fish and Shellfish Immunology 2:33-42.
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Galeotti, M., D. Volpatti, and M. Rusvai. Mitogen induced in vitro stimulation of
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Grimm, A. 5. 1985. Suppression by cortisol of the mitogen-induced proliferation of
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in M.J. Manning, and M. Tatner, editors. Fish Immunology. London, England.
Hamers, R. 1994. Studies on carp lymphoid cells from kidney, peripheral blood and
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European Association of Fish Pathologists 14:191-194.
Hardie, L. J., M. J. Marsden, Y. C. Fletcher, and C. J. Secombes. 1993. In vitro addition
of vitamine C affects rainbow trout lymphocyte responses. Fish & Shellfish
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Jeney, G., M. Galleotti, and D. Volpatti. 1994. Effect of immunostimulation on the
nonspecific immune response of sea bass, Dicentrarchus labrax L. Proceedings
International Symposium on Aquatic and Animal Health. Seattle, Washington.
Kaattari, S. L., and M. A.Yui. 1987. Polyclonal activation of salmonid B lymphocytes.
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Kaattari, S. L., and N. Holland. 1992. The one-way mixed lymphocyte reaction. Pages
61-66 in J. S. Stolen, T. C. Fletcher, D. P. Anderson, S.L. Kaattari, and A. F.
Rowley, editors. Techniques in Fish Immunology, Vol. 2. Fair Haven, New
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Leith, D. J., S. Holmes, S. Kaattari, M. Yui, and T. Jones. 1984. Effects of vitamin
nutrition on the immune response of hatchery-reared salmonids. Annual report to
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Le Morvan-Rocher, C., D. Troutaud, and P. Deschaux. 1995. Effects of temperature on
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LoPresto, C. J., L. K. Schwarz, and K. G. Burnett. 1995. An in vitro culture system for
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Luft, J. C., W. Clem, and J. E. Bly. 1991. A serum-free culture medium for channel
catfish in vitro immune responses. Fish and Shellfish Immunology 1:3l-139.
Milston, R. H., A. T. Vella, T. L. Crippen, M. S. Fitzpatrick, J. C. Leong, C. B. Schreck.
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Chinook salmon (Oncorhynchus tshawytscha) using flow cytometry. Fish and
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Wilkins and M. W. Sauders, editors. Biology and Management of Sablfish
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30
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31
CAPTURE-RELATED STRESSORS IMPAIR SABLEFISH, ANOPLOPOMA
FIMBRIA, IMMUNE SYSTEM FUNCTION
Ahsfrcict
Sablefish, Anoplopornafimbria, is a valuable North Pacific Ocean species that,
when not targeted in various commercial fisheries, is often a part of discarded bycatch.
Predictions of the survival of discarded fish are dependent on understanding how a fish
responds to stressful conditions. Our objective was to describe the immunological health
of sablefish exposed to capture stressors. In laboratory experiments designed to simulate
the capture process, we subjected sablefish to various stressors that may influence
survival: towing in a net, hooking, ecologically relevant temperature of seawater and air,
and air exposure time. Following the imposition of stress, the in vitro mitogen-stimulated
proliferation of sablefish leukocytes was used to assess the function of the immune
system in an assay we validated for this species. The results demonstrated that the
proliferative response of leukocytes from stressed sablefish, regardless of fishing gear
type, exposure to elevated seawater temperatures or air times, had significantly
diminished responses to the T cell mitogen concanavalin A or the B cell mitogen
lipopolysaccharide. A significant difference in the immunological response associated
with seawater or air temperature was not detected in sablefish. The duration and severity
of the capture stressors applied in our study were harsh enough to induce significantly
elevated levels of cortisol and glucose, but there was no difference in the magnitude of
levels among stress treatments. These data suggest that immunological suppression
occurs in sablefish subjected to capture-related stressors. The proposal that the immune
system is functionally impaired after exposure to capture-related stress suggests a
32
potential reason why delayed mortality is possible in discarded sablefish. Further studies
are needed to determine if delayed mortality in sablefish discards can be caused by
increased susceptibility to infectious agents resulting from stress-mediated
immunosuppression.
Introduction
The unaccounted mortality associated with the capture and release of non-targeted
fish to the ocean as discarded bycatch is one of the primary issues currently affecting
commercial fisheries management. Managers need to account for the mortality of
discarded fish to accurately assess fish populations and harvest impacts, yet discard
mortality rates of specific fisheries are generally unknown (Chopin et al. 1995; Pascoe
1997; NMFS 2003). Sablefish, Anoplopoma Jimbria, is a valuable North Pacific Ocean
species caught in several commercial fisheries and is often discarded due to size or catch
limits. Sablefish are susceptible to overexploitation due to their long lives with ages over
50 years regularly recorded (Kimura et al. 1983; McFarlane and Beamish 1983) and their
high migratory nature (Maloney and Heifetz 1997; Rutecki and Varosi 1997) with
distribution from northern Mexico to the Gulf of Alaska, westward to the Aleutian
Islands, and into the Bering Sea (Wolotira et al. 1993). Sablefish abundance is dependent
on strong year classes (McFar!ane and Beamish 1992) with juveniles providing the
primary source of recruitment into the sablefish fishery (Sigler et al. 2001). Juvenile
sablefish are very susceptible to trawl fisheries during near-shore residency and their
incidental discard can adversely effect sablefish recruitment (Sampson et al. 1997; Sigler
33
et al. 2001). The size-selective price structure of the commercial industry and the
minimum size limits also result in the discard of smaller fish potentially leading to greater
discard mortality (Schirripa and Methot 2003; Davis and Parker 2004).
Delayed mortality is one source of undetected mortality in discarded fishes.
Studies of fish held in the laboratory or field after capture-related stress, have indicated
that discard delayed mortality can vary with species of fish and the presence of physical
injury (Davis 2002). For example, in laboratory studies where fish were held for 60 days,
delayed mortality has been observed up to 30 days in halibut, Hippoglossus stemolepis,
14 days in walleye pollock, Theragra chalcogramma, and 35 days in sablefish with
physical injury (Davis 2002; 2004 in review). The observance of delayed mortality in
discards indicates that capture stressors may induce physiological modifications that
result indirectly in future mortality from predation, stress, and/or disease (Davis 2002).
Fish, like other vertebrates, respond to stressful stimuli through a neuroendocrine
cascade resulting in physiological and behavioral responses. After the stressor is
perceived, a physiological resistance phase follows leading to adaptation or
compensation. If the stress is overly severe and enduring, compensation may not be
feasible and exhaustion leads to mortality (Selye 1950; Schreck 2000). Situations that
push physiological systems beyond homeostasis can result in what Sterling and Eyer
(1998) have defined as allostasis: the ability of an organism to increase or decrease vital
functions to achieve a new form of stasis (Sterling arid Eyer 1988; McEwen 1999). Yet,
the cumulative strain on the organism from maintaining this allostatic response to stress
can result in an allostatic load (McEwen and Stellar 1993). Over time, the maintenance
34
of allostasis reduces the organism's ability to maintain normal physiological functions
(Pruett 2003).
Predictions of the survival of discarded fish are dependent on understanding how
a fish responds to stressful conditions. The capacity of fish to respond to a stressor can
be measured by biological, physiological, and behavioral changes that demonstrate the
degree of stress expressed (Wedemeyer et al. 1990). Commonly measured stress
indicators in sablefish such as plasma cortisol, lactate, and glucose, though useful in
quantifying the stress response, have not been correlated directly with capture-related
mortality (Davis et al. 2001; Davis 2002)
Since the imposition of a stressor disrupts a fish's capability to maintain
immunological function, understanding the capacity of the immune system to function
after an acute bycatch stressor may be important in assessing the survival of discarded
fish and further understanding the mechanisms behind delayed mortality It is known that
stress can have long-lasting negative effects on the general health of mammals (Dhabhar
and McEwen 2001) and teleosts (for reviews see: Anderson 1990; Barton and Iwama
1991; Schreck 1996; Wendelaar Bonga 1997; Weyts et al. 1999). Correlations of
immunosuppression and disease outbreaks in fish have been demonstrated in various
laboratory settings (Anderson 1990).
The objective of this study was to examine, in the laboratory, the effects of non-
lethal capture-related stressors on the immune function of the sablefish. The proliferative
response of leukocytes to a mitogen was utilized, in an assay we previously developed
and validated for sablefish, as a method of quantifying immune functions. Further
comprehension of the effects of the interactions of capture and environmental stressors on
35
sablefish are critical for providing understanding of what controls delayed discard
mortality. Knowledge of the immune response after the imposition of capture-related
stressors may lead to a better understanding of why fish die after capture.
Methods and Materials
Fish collection and maintenance
Juvenile sablefish [20-40 mm total length (TL)] were collected with neuston nets
in the spring of 2001 about 50 km offshore of Newport, Oregon and reared at the Hatfield
Marine Science Center (HMSC) in circular tanks (2.0 m diameter, 0.8 m depth, 31, 401
volume) supplied with sand-filtered and UV sterilized flow through seawater (29-32°/
salinity, 8-11 °C, 02 >90% saturation) at a replacement rate of 101. min'. Fish were fed
satiation on palletized salmon food three times a week. During the second year, fish were
maintained at 3 0-40 fish per tank in larger circular tanks (4.5m diameter, 1.0 m depth,
159,041 volume) supplied with flow-through seawater (201. min', 29-32°/
salinity, 02
>90% saturation, 8°C) and biweekly fed a diet of squid, Loligo opalescens, until satiated.
Experimental procedure
In laboratory experiments designed to simulate the capture process, we
investigated the impairment of immune system function in 2 year-old non-reproductive
sablefish. Each experiment involved three unstressed fish remaining at the maintenance
I'1
temperature of 8°C as a negative control and three fish subjected to a stressor. The trials
were replicated one week later. All experiments were conducted over a 4 month period at
HMSC in Newport, Oregon.
To simulate the stress imposed by trawling in a net and exposure to air that would
arise on a commercial vessel, sablefish (mean TL 36.8 cm) were transferred from the
maintenance tank by rapid netting to a tank with towing nets as described in 011a et a!
(1997). Briefly, to simulate the cod-ends of trawis, two 2.5 cm nylon diamond mesh nets
(1.2 m length, 0.7 diameter) were attached to rotating arms in a large tank (4.5 m
diameter, 1 m depth). For each trial, three fish were sequestered in one net and towed for
two hours at 1.1 mIs, a speed at which sablefish could not swim (011a et al. 1997) and at 8
°C, the temperature at which the fish were reared. Directly following the tow, the fish
were transferred in a net to a large basin (90 x 60 x 30 cm) in a temperature-controlled
room and exposed to 15 mm of air at 10 or 16°C.
The effects of hooking for two hours followed by exposure to 15 mm of air at 10
or 16 °C were determined in non-reproductive 2 year-old sablefish (mean TL 36.6). The
fish were transferred from the maintenance tank by rapid netting to a circular tank (2.0 m
diameter, 0.8 m depth, 8 °C) and hooked through the upper jaw onto commercial long-
line gear as described in Davis et al. (2001). Fish were maintained on the lines for two
hours, unhooked by hand and treated as described above to air exposures. Sablefish that
did not remain hooked on the commercial line for the entire two hours were noted
accordingly.
We simulated the capture of sablefish by trawling and exposure to warmer
temperatures during gear retrieval by towing non-reproductive 2 year-old sablefish (mean
37
TL 45.1 cm) for two hours followed by exposure to elevated seawater temperatures.
After towing as described above, the fish were transferred by rapid netting to a tank (3.0
m by 1.0 m), containing seawater temperatures of 10°C or 16°C for 15 minutes.
To simulate the stress imposed by air exposure on the deck of a fishing vessel,
sablefish (mean TL 36.8 cm) were transferred by dipnet from the maintenance tank into a
large basin (90 x 60 x 30 cm) in a temperature-controlled room. The fish were exposed
to air at 10 or 16 °C for either 15 or 30 mm and then placed back into a recovery tank
with 8°C seawater (3.0 m by 1.0 m) for 2 h or 1 h 45 mm, respectively. The fish were
placed into the recovery tank after air exposure to assure that the interval between the
onset of stress and the immune function measurement were equivalent for all laboratory
experiments.
After treatments were performed as described above, the fish were immediately
placed in a lethal dose (400 ppm) of tricaine methanesulfonate (MS-222). The total
length (TL) of each fish was recorded before blood and pronephros samples were
obtained from each specimen. Control fish from the maintenance tank (8 °C) were
sampled concurrently with the treatment groups.
Blood was collected from the caudal
vein with 3 mL heparinized vacutainers, the plasma separated by centrifugation and
stored at 80 °C until analyzed for cortisol and glucose levels. Plasma cortisol was
assayed according to radioimmunoassays techniques by Redding et al. (1984) and
validated for sablefish by 011a et al. (1997) and glucose was measured using a NOVA
analyzer.
Individual pronephros were harvested into tissue culture media (TCM) under
aseptic technique and lymphocytes isolated according to modified methods of Ellsaesser
and Clem (1986), Arkoosh et al. (1994), Milston et al. (2003), and Misumi (2004).
Immune function was assessed by quantifying the ability of pronephric leukocytes to
proliferate upon in vitro stimulation with the B cell mitogen lipopolysaccharide (LPS) or
the T cell mitogen concanavalin A (Con A) in an assay we previously validated for
sablefish (Lupes chapter 1 2004). Pronephric leukocytes from each sablefish were
cultured in triplicate with or without each mitogen. Proliferation was assessed by
leukocyte [3HJ-thymidine incorporation measured as counts per minute (cpm) by a
Wallac liquid scintillation counter.
Data analysis
Since each fish was sampled individually, each individual is an independent unit
and therefore considered the unit of measure for statistical purposes. Immune assays
were performed in triplicate. Therefore, data were derived from triplicate determinations
for each fish and individual proliferative responses were derived after the calculated
mean of triplicate determinations for the mitogen-stimulated cultures and the non-
stimulated cultures. Depending on the assay, in approximately 0.03% of the cases,
inclusion of an outlier would significantly affect the calculated stimulation index (SI)
according to a Wilcoxon rank-sum test and was disregarded, and the mean was therefore
calculated according to duplicated cultures. The proliferative responses were assessed for
each individual fish as SI calculated as the ratios of the mean [3HJ-thymidine uptake of
the mitogen stimulated cultures divided by the mean [3H]-thymidine uptake of the nonstimulated cultures.
For each capture-related laboratory experiment, the medium ± standard error (SE)
was calculated per experimental group. The data were analyzed with non-parametric
tests as the data did not fit a normal distribution and variances were not equivalent, and
calculated with SigmaStat software. A Kruskal-Wallis one-way analysis of variance
(ANOVA) of ranks test was utilized to observe any differences due to treatments,
followed by a Dunn's multiple-range test to examine differences between the median
values at different levels of capture. Differences were considered statistically significant
when P< 0.05.
Results
We studied the functional capability of pronephric leukocytes from sablefish
subjected to various capture-related stressors to respond in vitro to the B cell mitogen
LPS or the T cell mitogen Con A. Cells from control fish (unstressed at 8 °C) were
capable of responding to LPS and Con A (SI of 5.0 and 6.0 respectively), while the
proliferative responses were significantly depressed (P< 0.001) in fish that were towed
for 2 h at 8 °C followed by 15 mm of air at 10 or 16 °C (Figure 5). A significant
difference in the immunological response associated with air temperature was not
detected in sablefish exposed to 15 mm of air at 10 or 16 °C (P>0.5).
In comparison to
control fish, sablefish that were hooked for 2 hours and then transferred to air at 10 or 16
°C for 15 mm also presented significantly diminished (P<0.001) levels of leukocyte
proliferation upon in vitro stimulation with LPS and Con A (Figure 5). No significant
0
LPS
ConA
LPS
ConA
0
E
Cl)
4
Figure 5: Mean (+SEM) stimulation indices (SI) of sablefish subjected
°C comparison
to or 16 in
or towing at 8 °C, followed by 15 mm air exposure
at 10
controls (unstressed at 8 °C). The SI of stressed fish was significantly lower than in
controls denoted (*) (P <0.001).
41
difference was discerned between the 10 or 16 °C air temperature following the hook
stress for either one of the mitogens (P>0.05).
SI of sablefish towed for 2 hours at 8 °C followed by a 15 mm exposure to an
elevated seawater temperature of 10 or 16 °C were significantly (P< 0.001) diminished in
comparison to controls for both LPS and Con A (Figure 6).
A significant difference in
the immunological response associated with seawater temperature was not detected in
sablefish exposed to 15 mm at 10 or 16 °C. Sablefish that were directly exposed to air
temperatures of 10 or 16 °C for 15 or 30 mm and then placed in a recovery tank with 8 °C
seawater also presented a loss of mitogen responsiveness. The SI for LPS and Con A
were significantly reduced (P<0.05) in comparison to the control fish (unstressed at 8 °C)
(Figure 7). However, the proliferative responses did not vary significantly (P>0.05) due
to air temperature or minutes of air exposure.
Physiological variables measured directly after each treatment indicated that the
sablefish were undergoing significant stress responses. Cortisol plasma levels increased
significantly (P<0.001) from a median of 17.95 ng/ml in control fish (unstressed at 8 °C)
up to a median of 170.8 nglml in fish towed and held in air at 10 °C and to a median of
192.4 ng!ml in fish towed and held in air at 16°C (Figure 8). Fish towed and exposed to
elevated air temperatures of 10 or 16°C had median cortisol levels of 220.9 ng/ml and
176.4 nglml, respectively. In comparison to the controls, cortisol plasma levels were also
significantly elevated (P<0.001) in sablefish exposed to various air temperatures and
times and in fish towed and exposed to elevated seawater temperatures (Figure 8).
However, there were also no significant differences in the magnitude of cortisol levels
between the different treatments (Figure 8). Regardless of capture stressor, the cortisol
42
0
12
LPS
ConA
Figure 6: Mean (+SEM) stimulation indices (SI) of sablefish towed for 2 h at 8 °C
followed by 15 mm. exposure to an elevated seawater temperature of 10 or 16 °C in
comparison to controls (untowed at 8 °C). The SI of stressed fish was significantly lower
than in controls denoted (*) (P <0.001).
43
6
4
C
CID2
LPS
ConA
Figure 7: Mean (+SEM) stimulation indices (SI) of sablefish subjected to different air
exposure times (15 or 30 mm) and air temperatures (10 or 16°C). The SI of stressed fish
was significantly lower than in controls denoted (*) (P < 0.00 1).
44
Figure 8: Mean (+SEM) plasma cortisol concentrations (ng/ml) of sablefish subjected to
various stressor treatments in comparison to controls (unstressed at 8 °C): towed or
hooked for 2 h at 8°C, followed by 15 minute air exposure at 10 or 16 °C (a); towed for 2
h at 8 °C followed by 15 mm exposure to an elevated seawater temperature of 10 or 16
°C (b); different air exposure times (15 or 30 mm) and air temperatures (10 or 16°C) (c).
300
250
200
150
100
50
0
control
Tow
10°C
Tow
16°C
Hook
10°C
Hook
16°C
300
250
200
E-
ISO
0
0
100
C-)
50
0
control
16°C
10°C
300
250
200
150
C_)
100
50
0
control
Figure 8
15mm
30mm
15mm
3Omin
10°C
10°C
16°C
16°C
levels did not vary significantly (P>0.05) due to air temperature or minutes of air
exposure. Fish towed and exposed to elevated air temperatures of 10 or 16°C had
median blood glucose levels of 165.0 mg/ml and 158.5 mg/mi, respectively (Figure 9).
Fish captured by hooking and exposure to elevated air temperatures of 10 or 16°C had
median blood glucose levels of 114.0 mg/mi and 121.5 mg/mi, respectively (Figure 9).
Blood glucose levels significantly increased from a median of 50.0 mg/ml to elevated
glucose levels immediately after the induced stress, and were not significantly different
between capture by tow or hook (P > 0.05) (Figure 9). In comparison to the controls,
glucose levels were also significantly elevated (P<0.001) in sablefish exposed to various
air temperatures and times and in fish towed and exposed to elevated seawater
temperatures (Figure 9). However, there were also no significant differences in the
magnitude of glucose levels between the different treatments. Regardless of capture
stressor, the glucose levels did not vary significantly (P>0.05) due to air temperature or
minutes of air exposure.
Discussion
Regardless of treatment, the duration and combination of capture and
environmental stressors in our study were severe enough to result in a significant
reduction in the ability of pronephric leukocytes from stressed sablefish to respond in
culture to B and T cell mitogens. Immunosuppression as a result of stress is frequently
associated with decreased disease resistance to opportunistic pathogens (Wendelaar
47
Figure 9: Mean (+SEM) glucose levels (mg/dl) of sablefish subjected to various stressor
treatments in comparison to controls (unstressed at 8°C): towed or hooked for 2 h at 8 °C,
followed by 15 mm air exposure at 10 or 16 °C (a); towed for 2 h at 8 °C followed by 15
mm exposure to an elevated seawater temperature of 10 or 16 °C in (b); different air
exposure times (15 or 30 mm) and air temperatures (10 or 16 °C) (c).
250
200
150
100
0
50
0
control
Tow
Tow
Hook Hook
10°C
16°C
10°C
16°C
250
200
150
100
0
50
0
control
16°C
10°C
250
200
150
100
0
50
0
control
Figure 9
15mm
30mm
15mm
10°C
10°C
16°C
30mm
16°C
Bonga 1997) and our study demonstrates the cellular basis at which captured and released
sablefish could be more susceptible to infectious diseases. The fact that the immune
system is functionally impaired after exposure to capture-related stress sheds some light
on a potential reason why delayed mortality is possible in discarded sablefish. As a fish
is discarded back into the ocean, their ability to perform at the whole organism level may
be diminished for an extended period of time. Changes in feeding, social interactions,
schooling, swimming ability, orientation, predator evasion, and mortality corresponded
with increased capture stress intensity in previous laboratory studies in sablefish, Pacific
halibut, walleye pollock, and lingcod, Ophodion elongatus (for review see: Davis 2002).
"Injury, infection and delayed mortality have been observed in Atlantic herring that
escaped from a trawl, walleye Pollock that were towed in a net, and in Pacific halibut that
were towed in a net or hooked" (Davis 2004 in review). Davis (2004 in review)
suggested that in his laboratory study with sablefish towed in a net, delayed mortality
resulted from external skin and fin infections at the site of injury.
Over time the cumulative strain from maintaining physiological adaptation or
allostasis reduces an animal's ability to sustain normal physiological functions, including
immunological functions (McEwen and Stellar 1993; Pruett 2003); this would also be the
case with fish. Therefore, the ability of the immune system to protect the fish from injury
is impaired and can predispose the fish to disease and eventual death. Continuous harsh
environmental conditions can add to the cumulative damage, which accelerates the
pathophysiological processes and results in mortality sooner (McEwen 1999).
Based on the available evidence there is little doubt that the imposition of a
stressor will result in altered immune responsiveness (Kusnecov and Rabin 1994;
50
Schreck 2000). Modulation of the immune system due to stressors has been
demonstrated in both mammals and fish. In studies designed to evaluate immune
function after the imposition of stress, deficiencies in both cell-mediated and humoral-
mediated responses have been observed. The decreased lymphocyte responsiveness to
mitogen stimulation, antigen stimulation, and a reduction in lymphocyte cytotoxicity are
a few of the deficiencies that have been demonstrated in fish (Wendelaar Bonga 1997).
The in vitro stimulation of fish lymphocytes from the peripheral blood, thymus,
pronephros, and spleen is one of the most commonly used methods to assess the function
of the cellular immune system (Luft et al. 1991; Faisal and Hargis 1992). The mitogeninduced proliferation of lymphocytes from the pronephros was used in this study as it can
be considered a reasonable first approximation to show that the immune system of a fish
is capable of functioning (Ellasesser and Clem 1986).
Depending on the type of stress, exposure to a stressor can have a profound effect
on the immune system resulting in either enhancement or suppression of immune
function (Barton and Iwama 1991; Dhabhar and McEwen 1999; Weyts et al 1999; Pruett
2003). The enhancement of the immune system in fish has been observed following an
acute stress (Demers and Bayne 1997; Ruis and Bayne 1997) and immunosuppression
has been observed as a consequence of a chronic stress (Yin et al. 1995; Bly et al. 1997).
Although this effect of stress on the fish immune system is generally accepted, variability
in the response of the immune system after stress has been reported in both mammals and
fish. The variability in immune responses after the imposition of stress can be attributed
to the use of different species, different cell populations and different immunological
assays (Weyts et al. 1998). Considerations of stressor influences on immune function
51
also need to account for the duration, intensity, and frequency of the stressor, as this will
influence the magnitude and the duration of the response to the stressor (Pruett 2003).
In our study immunosuppression was observed in sablefish that were towed or
hooked and then exposed to air or increased temperatures. An undetectable significant
difference in immune responses between the two capture methods suggests that the two
gear types are equally damaging to the ability of leukocytes to proliferate. In these
experiments sablefish were exposed to two sequential stressors for a cumulative duration
of 2.25 or 2.5 hours. The combinations of sequential stressors used were nonlethal,
resulting in no immediate mortality or delayed mortality in sablefish during previous
capture-related laboratory experiments (Davis 2004 per comm.). Nonlethal conditions
were employed to avoid dealing with samples from morbid individuals and to ensure
consistency in sample sizes.
We assayed sablefish leukocytes directly after the imposition of bycatch stressors
and demonstrated that the immune system of sablefish is initially compromised directly
following capture-related stressors. However, there can also be a large range of immune
responses from suppression to enhancement within a session of stress exposure and/or the
time elapsing post-stress as the immune system recovers or fails to adapt to the stressor
(Maule et al. 1989; McEwen 1999; Ortuno 2001). The interval between the stress and the
immune function measurement are important; the timing of the immunological assay has
a large effect on the evaluation of the immunological endpoint (Cupps and Fauci 1982).
For example, in mice exposed to an auditory stressor there is a significant depression in
mitogenic responsiveness for the first two weeks, followed by a significant increase in
immune function response for the next two weeks (Borysenko and Borysenko 1982).
52
Maule et al. (1988) indicated that the fish immune response to a stressor operates similar
to mammalian systems in which there is a rapid immune depression, followed by a
transient immunostimulation, and ending in a more chronic suppression of the immune
function.
There is little information on the immunological response of fish to exposure to
two or more sequential stressors (Schreck 2000). Schreck (2000) presented a conceptual
physiological stress response model to sequential stressors suggesting that if the stressors
occur temporally close together, then there can be cumulative effects. Schreck's (2000)
model could explain why the combination of bycatch stressors, though possibly
considered acute in nature, resulted in a suppressive immune response rather than an
immune function enhancement. The cumulative effect of stress is apparent in other
laboratory studies in sablefish, Pacific halibut, walleye pollock, and lingcod, where stress
increased with stressor intensity as measured by changes in feeding, orientation, predator
evasion, and mortality (011a et al. 1997, 1998; Davis et al. 2001; Davis and 011a 2001,
2002; Davis 2004 in review). In these studies the initial capture (towing or hooking)
caused stress, which was then magnified by the addition of environmental stressors such
as air or increased seawater temperature, resulting in impaired behavior and increased
mortality. It was therefore surprising in our study that at treatment seventies that were
just below lethal levels, increased environmental air or seawater temperatures did not
cause further immunosuppression. Previous sablefish studies demonstrated that exposure
to above-ambient seawater temperature after simulated capture at 8 °C accentuated lactate
and potassium levels in sablefish (011a et al. 1998, Davis et al. 2001). Increased levels of
physiological stress and mortality have been observed to increase under field conditions
53
of increased temperature (Barton and Iwama 1991). Various studies also show that an
abrupt change in temperature can induce immune suppression in both specific and
nonspecific immunity (Ellis 1981, Wendelarr bonga 1997). Because we did not quantify
immune function after each successive stressor, we do not know if the initial capture
stress depressed the immune system so severely, not allowing for further
immunosuppression induced with each sequential stressor. However, we cannot
disregard the possibility that the sequence of stressors even at nonlethal seventies were
debilitating to the immune system due to the cumulative effect of stress. Given the
different natures of the stress stimuli in our study and the complexity of the immune
response, it is difficult for us to classify our sequence of stressors as a combination of
acute stressors or a cumulative chronic stressor simply based on the immune response
observed.
Sablefish directly exposed to air exposure for 15 or 30 minutes also did not reflect
the general acute immunological response to a stressor, resulting instead in decreased
lymphocyte mitogen-induced proliferation. However, these results are consistent with
other studies that have demonstrated that non-lethal aerial emersions of salmonids even
as brief as 30 seconds (Maule et al. 1988) caused a significant depression in immune
function. Many nonlethal acute stressors including confinement, crowding, acute
handling, transport, and increased water temperature can also induce immunosuppression
(Wendelaar Bonga 1997). Sublethal levels of stress produced by hypoxia have also
demonstrated impairment in immune function (Mellergaar and Nielsen 1995; Ortuno et
al. 2002).
in previous experiments, sablefish mortality was only observed after 30
minutes in air and increased dramatically between 30 and 50 mm (Davis and Parker
54
2004). Davis and Parker (2004) also demonstrated that there was behavioral impairment
after just 10 minutes in air possibly reflecting physiological injury.
The lack of air temperature effects on the immune function was consistent with
lack of air temperature effects on mortality (Davis and Parker 2004). Davis and Parker
(2004) suggested that the warming of sablefish is not very efficient in air and therefore
body core temperature remains low. The warming of sablefish is more efficient in water
with smaller fish reaching higher body core temperature more quickly than larger fish
(Davis et al. 2001). One possible explanation for the lack of air temperature effect on
immune function is that the direct exposure to the air for 15 or 30 minutes so severely
depressed the immune system such that further immunosuppression was not possible in
sablefish subjected to increases in air temperature.
Our study found suppressed immune function in sablefish subjected to capturerelated stressors, but we do not know the mechanisms underlying the immune
suppression. How the function of lymphocytes, macrophages and other cells of the
immune system are modified by the duration and frequency of the stressor are not well
understood (Krusnecov and Rabin 1994). Prolonged stress exposes lymphocytes to
continuously elevated levels of hormones, costicosteriods, catecholamines (CA) and
other neuropeptides from lymphoid organs. The stress-related immune dysfunction
appears to be driven by these neuroendocrine responses with glucocorticoids as the major
mediators of immunosuppression (Pickering 1981; Adams 1990; Wendemeyer et al.
1990; Wendelaar Bonga 1997; McEwen and Seeman 1998; Pruett 2003).
The duration and severity of the capture stressors applied in our study were harsh
enough to induce significantly elevated levels of cortisol and glucose. However, there
55
was no difference in the magnitude of plasma cortisol and glucose among stress
treatments. Davis et al. (2001) observed greater plasma cortisol, lactate, and potassium
levels in towed sablefish followed by elevated temperature and air than in hooked
sablefish followed by elevated temperature and air. However, in that study the stressinduced capture was inflicted upon sablefish for a total of 4 hours, two times the duration
of our study. The greater duration of capture stress in their study possibly contributed to
further increases in cortisol and glucose levels observed after the towing stress.
Our study demonstrates that the sablefish immune system is functionally
impaired after exposure to capture-related stressors. As a fish is discarded back into the
ocean, their ability to perform at the whole organism level may be diminished for an
extended period of time. Stress relocates metabolic energy from performance activities
such as growth, reproduction and immune responses towards activities required to restore
homeostasis such as cardiac output, oxygen uptake, and hydromineral balance (Barton
and Iwama 1991). Thus, the energetic costs associated with adapting and recovering
from the stressor are significant and reduce the performance capacity of the fish (Scheck
1981). Over time the cumulative strain on a discarded fish from maintaining
physiological adaptation reduces its ability to sustain normal immunological functions.
Therefore, the ability of the immune system to protect the fish from damage is impaired
and can predispose the fish to disease and eventual death. However, further studies are
needed to determine if delayed mortality in sablefish discards can be caused by increased
susceptibility to infectious agents resulting from stress-mediated immunosuppression.
56
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McFarlane, G.A. and R.J. Beamish 1992. Climatic influence linking copepod production
with strong year-classes in sablefish, Anoplopomafimbria. Canadian Journal of
Fisheries Aquatic Science 49:743-753.
Mellergaard, S. and B. Nielsen. 1995. Impact of oxygen deficiency on the disease status
of common dab Lima limanda. Diseases of Aquatic Organisms 22: 101-114.
Milston, R. H., A. T. Vella, T. L. Crippen, M. S. Fitzpatrick, J. C. Leong, C. B.
Schreck. 2003. In vitro detection of functional humoral immunocompetence in
juvenile Chinook salmon (Oncorhynchus tshawytscha) using flow cytometry. Fish
and Shellfish Immunology 15:145-158.
Misumi, I. 2004. Immune response ofjuvenile chinook salmon (Oncorhynchus
tshawytsha) to p'p'-DDE and tributyltin. M.Sc. thesis, Oregon State University,
Corvallis, Oregon.
National Marine Fisheries Service (NIMFS). 2003. Evaluating bycatch: a national
approach to standardized bycatch monitoring programs. NOAA, NMFS, Silver
Springs, Maryland.
011a, B.L., M. W. Davis, and C. B. Schreck. 1997. Effects of simulated trawling on
sablefish and walleye pollock: the role of light intensity, net velocity, and towing
duration. Journal of Fish Biology 50:1181-1194.
011a, B.L., M. W. Davis, and C. B. Schreck, C.B. 1998. Temperature magnified
postcapture mortality in adult sablefish after simulated trawling. Journal of Fish
Biology 53:743-75 1.
Ortuno, J. M., A. Esteban, and J. Meseguer. 2002. Lack of effect of combining different
stressors on innate immune responses of seabream. Veterinary Immunology and
Immunopathology 84:17-27.
59
Parker, S. J., P. S. Rankin, R. W. Hannah, and C. B. Schreck. 2003. Discard mortality of
trawl-caught lingcod in relation to tow duration and time on deck. North
American Journal of Fisheries Management 23:530-542.
Pascoe, S. 1997. Bycatch management and the economics of discarding. Food and
Agricultural Organization of the United Nations. Fisheries Technical Paper, No.
370.
Pickering, A. D. 1981. Introduction: the concept of biological stress. pp 1-9 in A. D.
Pickering, editors. Stress and Fish. Academic Press. New York.
Pruett, S. B. 2003. Stress and the immune system. Pathophysiology 9:133-153.
Ruis, M. A.W., and C. J. Bayne. 1997. Effects of acute stress on blood clotting and
yeast killing by phagocytes of rainbow trout. Journal of Aquatic Animal Health
190-195.
Rutecki, T. L., and E. R. Varosi. 1997. Distribution, age, and growth ofjuvenile sablefish
Anoplopomafimbria, in Southeast Alaska. Pages 45-54 in M.E. Saunders and
M.W. Wilkins, editors. Biology and management of sablefish. U.S. Department
of Commerce, NOAA Technical Report NMFS-130.
Redding, J. M., C. B. Schreck, E. K. Birks, and R. D. Ewing. 1984. Cortisol and its
effects on plasma thyroid hormone and electrolyte concentrations in fresh water
and during seawater acclimation in yearling coho salmon, Oncorhynchus kisutch.
General Comparative Endocrinology 56:146-155.
Sampson, D.B., W. Barass, M. Saelens and C. Wood. The bycatch of sablefish,
Anoplopomafimbria, in the Oregon Whiting Fishery. Pages 183-194 in M.E.
Wilkins and M.W. Saunders, editors. Biology and Management of Sablefish
Anoplopoma fimbria. U.S. Department of Commerce, NOAA Tech. Report.
NMFS-1 30.
Schirripa, M. J., and R. D. Methot. 2002. Status of the sablefish resource off the U.S.
Pacific coast in 2001. Pacific Fishery Management Council, Seattle, Washington.
Schreck, C.B. 1981. Stress and compensation in teleostean fishes: response to social and
physical factors. Pages 295-32 1 in A.D. Pickering, editors. Stress and Fish.
London.
Schreck, C.B. 1996. Immunomodulation: endogenous factors. in G. Iwama and T.
Nakanishi, editors. The Fish Immune System. Vol. 15 'Fish Physiology',.
Academic Press, San Diego, California.
Schreck, C. B. 2000. Accumulation and long-term effects of stress in fish. Pages 147158 in G. P. Moberg and J. A. Mench, editors. The Biology of Animal Stress.
CABI Publishing, Oxon, U.K.
Selye, H. 1950. Stress and the general adaptation syndrome. British Medical Journal
1(46670): 1383-1392.
Sigler, M.F. T. L. Rutecki, D.L. Courtney, J. F. Karinen, and M. S. Yang. 2001. Young
of the year sablefish abundance, growth, and diet in the gulf of Alaska. Alaska
Fishery Research Bulletin 8:57-70.
Spenser, M. 2000. Behavioral and physiological indicators of stress in sablefish
(AnoploomajImbria). M.Sc. thesis, Oregon State University, Corvallis, Oregon.
Sterling, P., and J. Eyer. 1988. Allostasis: a new paradigm to explain arousal pathology.
Pages 629-649 in Fisher, S. and Reason, J., editors. Handbook of Life Stress,
Cognition and Health. John Wiley & Sons, New York, NewYork.
Wedemeyer, G. A., B. A. Barton, and D. J. Mcleay. 1990. Stress and acclimation. Pages
45 1-489 in C. B. Schreck and P. B. Moyles, editors. Methods for Fish Biology.
American Fisheries Society, Bethesda, Maryland.
Wendelaar Bonga, S. E. 1997. The stress response in fish. Physiological Reviews 77:
59 1-625.
Weyts. F.A.A., N.Cohen, G. Flik, and B.M.L. Verburg-van Kemenade. 1999.
Interactions between the immune system and the hypothalamo-pituitary-interrenal
axis in fish. Fish and Shellfish Immunology 9:1-20.
Wolotira, R. J., T. M. Sample, S. F. Noel, and C. R. Iten. 1993. Geographic and
bathymetric distributions for many commercially important fish and shelifishes
off the west coast of North America, based on research survey and commercial
catch data, 1912-1984. NOAA Tech. Memo. NMFS-AFSC-6. ppl84.
Yin, Z., T. J. Lam, and Y. M. Sin. 1995. The effects of crowding stress on the nonspecific immune response in fancy carp (Cyprinus carpio L.). Fish and Shellfish
Immunology 5:519-529.
61
CONCLUSION
The present study demonstrated that pronephric leukocytes are suitable for use in
in vitro assays to evaluate the proliferative responses of sablefish cells to classic
mammalian B and T cell mitogens. The development of an in vitro culture system for
sablefish leukocytes will allow for future assessment of the function of the sablefish
cellular immune system in aquaculture and natural populations. This immunological
assay should be usable at sea for evaluation of fish health and assessment of the effects of
fishing tactics and gear. Use of this assay to assess and measure the sablefish immune
response to laboratory-simulated bycatch conditions leads to further insights regarding
delayed discard mortality. The functional impairment of the immune system after
exposure to capture-related stress proposes a potential reason why delayed mortality is
possible in discarded sablefish. As fish are returned to the ocean, their ability to perform
at the whole organism level may be diminished for an extended period. Over time the
cumulative strain on a fish from maintaining physiological adaptation reduces its ability
to sustain normal immunological function. Therefore, the ability of the immune system
to protect the fish is impaired and can possibly predispose a discarded fish to disease and
eventual death. Consequently, following the imposition of nonlethal capture stressors,
morbidity is not sudden and mortality is not observed until weeks later, as reported in
previous field and laboratory experiments (Davis 2002; 2004 in review). However,
further studies are needed to determine if delayed mortality in sablefish discards can be
caused by increased susceptibility to infectious agents resulting from stress-mediated
immunosuppression.
62
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