Are hsps suitable for indicating stressed states in fish?
1 Institute for Marine Biosciences, National Research Council of Canada,
1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1
2 The University of British Columbia, Faculty of Agricultural Sciences,
Vancouver, British Columbia, Canada, V6T 1Z4
* Author for correspondence (e-mail: george.iwama{at}nrc.edu)
Accepted 8 September 2003
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Summary |
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Key words: cortisol, fish, heat shock protein, stress, stressor
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Introduction |
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Fish also respond at the cellular level to stressors. This response
comprises a suite of protein changes that includes the increased synthesis of
heat shock proteins (hsps; Iwama et al.,
1998). Our understanding of the cellular stress response in fish
has also increased substantially (Basu et
al., 2002
). In addition to the variables mentioned above, the
cellular stress response can vary according to tissue and hsp family. Several
studies have attempted to establish a relationship between the physiological
and cellular stress responses, but there exist apparent inconsistencies
between these two levels of response. Thus, a fish that may present a
physiological response to a stressor may not show any change in cellular hsp
profile.
The definition and discussion of stress often defaults to taking on a negative perspective. The connotation of the word in common use is generally negative. As experimental biologists, we are all involved in the practice of imposing some perturbation and measuring a biological response. The word or concept of 'stress' is commonly invoked when inexplicable or unexpected, and negative, results are obtained. This underscores the importance of clarity in defining this important response. Thus, indicators of the generalized stress response in fish are worthy of discussion.
Our goal in this brief review is to bring to light several factors that need be taken into account to use hsps as an indicator of a general state of stress in fish. Indeed, none of the current indicators of stress, including the stress hormones, are 100% suitable in reflecting stressed states in fish. We will argue that the use of hsps as indicators of generalized stress response is still premature.
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Stressors and the generalized stress response |
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The physiological stress response |
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The susceptibility of fish to different stressors also has genetic
components. There are differences in the stress responses among species (see
Vijayan and Moon, 1994) and
differences among stocks of the same species in their tolerance to applied
stressors (see Iwama et al.,
1999
).
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The cellular stress response |
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A complete understanding of the mechanisms underlying the sensing of a
stressor and the regulation of hsps is far from clear. Studies on hsp70, the
most extensively studied hsp, have demonstrated that the regulation of
hsp70 gene expression occurs mainly at the transcriptional level
(Fink and Goto, 1998).
Analysis of hsp genes and a comparison of heat shock regulatory
elements from a variety of organisms led to the identification of a
palindromic heat shock element (hse): CNNGAANNTTCNNG
(Bienz and Pelham, 1987
). It
has been demonstrated that hsp induction results primarily from the binding of
an activated heat shock transcription factor (hsf) to an hse upstream of
hsp genes (Morimoto et al.,
1992
). Recently, it has been shown in zebrafish (Danio
rerio) that the transcriptional regulation of hsp genes, in
response to heat shock, is also mediated by an hsf
(Rabergh et al., 2000
). Since
most of the hsp genes do not contain introns, the mRNA is rapidly
translated into nascent proteins within minutes of exposure to a stressor.
Genomic sequences for hsp70 are being elucidated in fish, including
rainbow trout (Oncorhynchus mykiss;
Kothary et al., 1984
), medaka
(Oryzias latipes; Arai et al.,
1995
), zebrafish (Lele et al.,
1997
), pufferfish (Fugu rubripes;
Lim and Brenner, 1999
) and
tilapia (Oreochromis mossambicus;
Molina et al., 2000
).
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Heat shock proteins as indicators of stress |
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It is possible that high-throughput genomic and proteomic technologies, accompanied by appropriate bioinformatics, will enable a more comprehensive profiling of the responses of the cell to stressors. An unbiased description of the protein changes that characterize the generalized response to stressors would contribute to a vital foundation upon which we could base future experiments.
The induction of various hsp families in fish has been reported in cell
lines, primary cultures of cells, as well as in various tissues from whole
animals (Iwama et al., 1999).
While the majority of these studies have focused on the various effects of
heat shock, there is increasing interest in the physiological and protective
role of hsps following exposure of fish to various environmental
stressors.
There have been several efforts to validate the use of the hsp response as
an indicator of stressed states in fish. Indeed, it has been shown that
several forms of environmental stressors can induce the hsp response in fish.
For example, increased levels of various hsps have been measured in tissues of
fish exposed to industrial effluents
(Vijayan et al., 1998),
polycyclic aromatic hydrocarbons (Vijayan
et al., 1998
), several metals such as copper, zinc and mercury
(Sanders et al., 1995
;
Williams et al., 1996
:
Duffy et al., 1999
),
pesticides (Hassanein et al.,
1999
) and arsenite (Grosvik
and Goksoyr, 1996
). These studies and others revealed that the use
of hsp as an indicator of stressed states in fish is a very complex issue. The
hsp response can vary according to tissue
(Rabergh et al., 2000
;
Smith et al., 1999
), distinct
hsp families (Smith et al.,
1999
) and stressor (Airaksinen
et al., 2003
), and the sensitivity of hsp expression can also vary
with the species (Basu et al.,
2001
; Nakano and Iwama,
2002
), developmental stage
(Santacruz et al., 1997
;
Lele et al., 1997
;
Martin et al., 2001
) and
season (Fader et al.,
1994
).
When studying stress in aquatic organisms it is very important to establish
whether experimental procedures such as handling, sampling and other physical
stressors are affecting the hsp response. While handling and sampling
procedures can affect common indicators of the physiological stress response
in fish, such as plasma cortisol levels, it has already been demonstrated in
rainbow trout that handling stress does not alter levels of hepatic hsp70
(Vijayan et al., 1997), and
levels of muscle, gill, heart and hepatic hsp60
(Washburn et al., 2002
).
Recently, Zarate and Bradley
(2003
) showed that common
forms of hatchery-related stressors (exposure to anesthesia, formalin,
hypoxia, hyperoxia, capture stress, crowding, feed deprivation and cold
stress) did not alter levels of gill hsp30, hsp70 and hsp90 in Atlantic salmon
(Salmo salar). While these studies demonstrated that common stressors
did not elicit an hsp response and therefore do not interfere with its use as
an indicator, they also showed that hsp may not be a sensitive indicator of
stressed states when physical stressors are applied in aquaculture
operations.
There are a few studies that relate the physiological and cellular stress
responses in vivo. In mammals, it is known that hsps are involved in
the immune response (Young,
1990; Breloer et al.,
2001
). Two studies have demonstrated increased levels of hsp70 in
various tissues in fish exposed to pathogens
(Forsyth et al., 1997
;
Ackerman and Iwama, 2001
). The
later study revealed that rainbow trout infected with a bacterial pathogen
(Vibrio anguilarum) increased levels of hsp70 in hepatic and head
kidney tissues prior to clinical signs of the disease. This study also showed
that the peak in hepatic hsp70 levels corresponded to that of plasma cortisol
levels, which occurred 5 days after the challenge. However, head kidney hsp70
levels increased significantly on the fourth day after challenge, when plasma
cortisol levels were similar to the control group.
Such interests in stress research at the organismal level have taken us to
the intertidal zone, where fish cope with changes to their environment, the
tidepool, that are unpredictable from one low tide to the next. We have been
working extensively on the functional importance of hsp70 in thermal tolerance
using two intertidal fishes, the tidepool sculpin (Oligocottus
maculosus) and fluffy sculpin (Oligocottus snyderi), as model
species. While both sculpins inhabit the intertidal zone along the west coast
of North America, the tidepool sculpin has a higher thermal tolerance than the
fluffy sculpin. As in research by Hightower et al.
(1999) on the desert pupfish
(Cyprinodon macularius), we have observed that the levels of
constitutive hsp70 and the scope for increase in hsp correlate with the
ability of the tidepool sculpins to cope with these environmental changes
(Nakano and Iwama, 2002
). The
tidepool sculpin, which is exposed to a wider range of fluctuation in water
temperature in upper tidepools, had higher constitutive liver hsp70 levels
that were only slightly influenced by changes in water temperature. However,
the fluffy sculpin, which prefers lower tidepools with smaller changes in
water conditions, showed a larger change in liver hsp70 levels in response to
thermal stress at lower temperatures. We suggested that the less thermally
sensitive sculpin might enhance its thermal tolerance by having a large
constitutive pool of hsp70. Therefore, in order to use the hsp level as a
realistic stress indicator in various fish species, it is essential to
understand such relationships between the organismal stress tolerance of fish
and the cellular stress response so that a better understanding of the
functional significance of hsps in natural populations of fish can be
obtained.
In addition to the differences that we have observed in the cellular stress
response of fish species with overlapping habitats, differences in the
cellular stress response of a particular fish species inhabiting different
geographical locations have been reported by Norris et al.
(1995). Furthermore, seasonal
variations in the hsp response within a species and between species of fish
have been shown by Dietz and Somero
(1992
) and Fader et al.
(1994
). It is also important
to consider that some fish species may not show a heat shock response. Hofmann
et al. (2000
) showed that
hsp70 is not induced by temperature stress in the Antarctic fish
Trematomus bernacchi. Thus, generalizations about the hsp response
cannot be made unequivocally, and more knowledge is needed in order to know
when one can use a specific hsp family as an indicator of stress in fish.
A good indicator of cellular mechanisms should also enable the cellular
response to be linked to effects at higher levels of biological organization
(Lewis et al., 1999). A few
recent studies have explored the possible direct relationship between the
physiological and cellular stress responses in fish. While we are far from
understanding this relationship, these studies have demonstrated that steroid
hormones, including cortisol, may have a direct influence on the cellular
stress response. Stress levels of plasma cortisol attenuated the heat
stress-induced increases of gill hsp30 in cutthroat trout (Oncorhynchus
clarki clarki; Ackerman et al.,
2000
), liver and gill hsp70 in rainbow trout, gill hsp70 in
tilapia (Basu et al., 2001
),
hsp90 mRNA in primary cultures of rainbow trout hepatocytes
(Sathiyaa et al., 2001
) and
hsp70 in primary cultures of rainbow trout hepatocytes
(Boone and Vijayan, 2002
).
Recently Basu et al. (2003
)
showed a functional and structural link between hsp70 and the glucocorticoid
receptor in rainbow trout. They also showed that the glucocorticoid receptor
heterocomplex contains hsp70 and that the association between hsp70 and the
glucocorticoid receptor can be altered according to the stressor.
Several stressors that can clearly elicit a physiological stress response
are also able to affect the cellular stress response in fish. However, the hsp
responses seem to vary considerably according to tissue, family of hsp,
organism, developmental stage and stressor. The complexity of the available
data makes it difficult to conclude that hsps are general indicators of the
stress response in fish. We suggest that if hsps are to be used as indicators
of the stress response, it will probably have to be done in a stressor- and
species-specific manner. Finally, there is a need to determine the hsp
response during periods of chronic stress. Even in terms of the
well-established physiological responses, little information is available for
persistent stressors. There is also a need to know how fish respond when
exposed simultaneously to multiple stressors or to sequential stressors
(Schreck, 2000). Further
research that will elucidate the relationship between the cellular and
physiological stress responses is needed. Our state of understanding of the
cellular stress response in fish precludes the simple use of hsps as
indicators of stress in fish.
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