Volume 50, Number 4 2009 387

Abstract
Fish and other aquatic animals are subject to a broad variety
of stressors because their homeostatic mechanisms are
highly dependent on prevailing conditions in their immedi-
ate surroundings. Yet few studies have addressed stress as a
potential confounding factor for bioassays that use sh as
test subjects. Common stressors encountered by captive sh
include physical and mental trauma associated with capture,
transport, handling, and crowding; malnutrition; variations
in water temperature, oxygen, and salinity; and peripheral
effects of contaminant exposure or infectious disease. Some
stress responses are detectable through gross or microscopic
examination of various organs or tissues; as reported in the
literature, stress responses are most consistently observed in
the gills, liver, skin, and components of the urogenital tract.
In addition to presenting examples of various stressors and
corresponding morphologic effects, this review highlights
certain challenges of evaluating stress in sh: (1) stress is an
amorphous term that does not have a consistently applied
denition; (2) procedures used to determine or measure
stress can be inherently stressful; (3) interactions between
stressors and stress responses are highly complex; and (4)
morphologically, stress responses are often difcult to dis-
tinguish from tissue damage or compensatory adaptations
induced specically by the stressor. Further investigations
are necessary to more precisely dene the role of stress in
the interpretation of sh research results.
Key Words: contaminant; crowding; sh; handling; histol-
ogy; nutrition; temperature; salinity; stress
Introduction
I
n 1936 a scientist named Hans Selye, upon observing
effects of noxious stimuli in laboratory animals, coined
the term stress and dened it as the non-specic re-
sponse of the body to any demand for change (Selye 1936,
Claudia Harper and Jeffrey C. Wolf
Claudia Harper, DVM, DACLAM, is Director of Preclinical at Amgen Inc.
Jeffrey C. Wolf, DVM, DACVP, is a toxicologic veterinary pathologist at
Experimental Pathology Laboratories Inc. in Sterling, Virginia.
Address correspondence and reprint requests to Dr. Jeffrey C. Wolf,
Experimental Pathology Laboratories, 45600 Terminal Drive, Sterling,
VA 20166 or email jwolf@epl-inc.com.
Morphologic Effects of the Stress Response in Fish
32). Subsequent researchers have proposed a variety of al-
ternate denitions, but, as commonly used, the word stress
still conveys a vague notion of unease, distress, discomfort,
or disturbance. Ambiguity exists in part because the word
can be used to indicate one of three different components of
what is essentially a cause and effect relationship: (1) a
physical or mental stimulus, (2) an individuals physical or
mental awareness of that stimulus, or (3) the individuals
physical or behavioral response to the stimulus. For exam-
ple, exposure to cold temperatures (stimulus) can make an
individual feel cold (awareness) and evoke shivering (re-
sponse); regarded independently, each of these elements
might be considered stress. As one frustrated scientist re-
portedly claimed, Stress, in addition to being itself and the
result of itself, is also the cause of itself (Roberts 1950,
105). To avoid confusion, we use the term stressors to in-
dicate stressful stimuli and stress responses to designate
the reactions to such stimuli.
The purpose of this article is to review morphologic ef-
fects of various stressors in sh as determined by gross or
histopathologic investigation. In addition, descriptions of
both tissue-specic and non-tissue-specic stress responses
are provided. For more general discussions of piscine stress,
several excellent reviews are available (Barton 2002; Gratzek
and Reinert 1984; Iwama et al. 2004a; Pickering 1981).
Stressors and Stress Responses
Throughout the animal kingdom, many types of stressors are
universal simply because the basic needs of most animals are
similar. Examples of universal stressors include deviations
from optimal ranges for environmental parameters (e.g., ambi-
ent temperature, oxygen supply), insufcient food availability,
inadequate refuge from sunlight or predators, and the demands
of social interactions such as territorial disputes. Other stre ss-
ors are unique to certain animal groups or habitats.
As compared to terrestrial inhabitants, sh and other
aquatic creatures are subject to a broader variety of stressors
because their homeostatic mechanisms are highly dependent
on prevailing conditions in their immediate surroundings.
Examples of additional stressors for sh include uctuations
in water salinity, pH, hardness, alkalinity, dissolved solids,
water level or current, and exposure to waterborne pathogens
or toxicants. Fish reared in connement systems often expe-
rience further pressures of crowding, handling, suboptimal
nutrition, and nitrogenous waste accumulation.
388 ILAR Journal
Evidence indicates that certain stress responses are well
conserved evolutionarily. In terms of behavior, an obvious
example is the instinctive urge to ght or ee when faced
with an adverse stressor such as predation. Many physiologi-
cal responses to stressors are also remarkably comparable
among taxonomically diverse animals. For instance, com-
mon among all vertebrates is the stressor-induced secretion
of adrenergic and glucocorticoid hormones; the latter espe-
cially is considered a hallmark of the stress response (Nesse
and Young 2000). Although sh lack adrenal glands per se,
analogous production and release of adrenal cortical and
medullary hormones occur in the interrenal cells and chro-
mafn tissues, respectively, both of which are typically lo-
cated in the piscine anterior kidney.
As in the case of so-called higher vertebrates, the secre-
tion of stress-related hormones in sh can be a double-edged
sword. The activities of these hormones are clearly bene-
cial when acute action and its consequences take priority, as
they elicit a heightened state of alertness, increase blood
pressure and respiration, promote hepatic glycogen catabo-
lism to provide a source of energy via glucose, and limit ex-
cessive tissue damage from inammatory reactions to trauma
or illness (Nesse and Young 2000). However, hormonal stress
responses that overcompensate or persist can also have nega-
tive effects, such as immune suppression, depletion of en-
ergy reserves, muscle breakdown, and, in sh, interference
with osmoregulation as a result of altered mineral metabo-
lism (Banerjee and Bhattacharya 1995).
Measurement of Stress Responses
Despite the commonality of the stress response, for several
reasons it is not always easy to measure its effects in an ex-
perimental setting. First, such responses are not all or noth-
ing events. As exposure to a particular stressor increases in
magnitude and duration, the outcome can progress from a
complete lack of clinical effects to relatively subtle manifes-
tations (e.g., decreased reproductive performance) to patent
signs of disease (e.g., life-threatening microorganism infec-
tions) (Benli et al. 2008). Further complicating this picture
are adaptive mechanisms that may compensate to varying
degrees for chronic or low-level stress and thereby contrib-
ute to inconsistency in stress responses among test subjects.
A second challenge is that, analogous to the observer effect
described in quantum physics, efforts to measure in vivo
stress responses can be stressful in and of themselves; for
example, the capture process can affect levels of measured
cortisol levels in wild sh collected for stress management
research (Cleary et al. 2002; Tsunoda et al. 1999). A third
challenge for scientists is that the effects of the stress response
can be difcult to distinguish from effects of the stressor it-
self (Selye 1955); this represents an important obstacle in
endocrine disruption research, in which it is necessary to dif-
ferentiate the particular effects of hormonally active sub-
stances from their concomitant ability to contribute to the
stress response (Norris 2000).
To date, methods for evaluating stress responses in sh
have involved a variety of endpoints:
whole body or organ weight measurements (e.g., condi-
tion factor, hepatosomatic index, and gonadosomatic in-
dex) (Dutta et al. 2005; Hosoya et al. 2007; Spencer et al.
2008);
biochemical assays (e.g., plasma cortisol, corticosterone,
glucose, tissue damage enzymes, and heat shock pro-
teins) (Acerete et al. 2004; Barton 2002; Dutta et al.
2005; Hosoya et al. 2007; Iwama et al. 2004b; Olsen
et al. 2008; Trenzado et al. 2008);
immune function (Choi et al. 2007);
gene expression patterns (Basu et al. 2001, 2002;
Marques et al. 2008; van der Meer et al. 2005);
measurement of sh steroids in water (Scott and Ellis
2007); and
macroscopic and microscopic anatomy (numerous refer-
ences cited in the following text).
As a research tool, the histopathologic evaluation of whole
body sections from small sh species offers numerous advan-
tages, including the ability to observe a wide variety of organ
systems in relatively few tissue sections, the ability to iden-
tify concurrent disease problems, the long-term stability of
the raw data (because histologic sections are mounted on
glass slides), and, perhaps most importantly, the ability to de-
tect treatment-induced changes that might otherwise remain
undiscovered.
Although the potential for stress responses to confound
certain experimental results can be high, only a limited num-
ber of studies have specically addressed the effects of such
responses on tissue histomorphology. For example, despite
documented stressor-induced alterations of reproductive sys-
tem endpoints (Cleary et al. 2002; Contreras-Snchez et al.
1998), there has been little effort to determine potential histo-
pathologic effects of the stress response (e.g., as modeled by
cortisol administration) on sh gonads or gonadal ducts; such
effects might include increased germ cell degeneration in the
ovary (oocyte atresia) and/or testis. Such ndings would be
signicant because those same types of changes are often re-
garded as prima facie evidence of endocrine disruption (Heiden
et al. 2006; Leino et al. 2005; Rasmussen et al. 2005). Sea-
sonal changes also are known to affect sh gonads and lead
to morphological changes (Abe and Munehara 2007).
Fish-Specic Stressors
There are roughly 30,000 known species of sh, and both
wild and captive shes occupy a remarkably diverse array of
habitats. Accordingly, environmental conditions that might
be optimal for one species are inherently stressful for an-
other. Given the number of potential stressors, and the fact
that sh may be exposed to multiple stressors simultane-
ously, the range of potential stress-inducing situations is al-
most limitless. This section provides brief descriptions of
commonly encountered stressors and the anatomic sites in
which corresponding morphologic effects tend to occur.
Volume 50, Number 4 2009 389
Specic histopathologic ndings are described in more de-
tail in the following section, categorizing stress responses by
tissue type or organ system.
Capture, Transport, and Handling
Capture, transport, and handling are obvious stressors for
captive sh, but wild sh may also experience these distur-
bances, for example through catch and release programs in
recreational sheries. Procedures that can intensify the stress
response in aquacultured sh include sorting, grading, and
vaccine administration (Burgess and Coss 1982). Additional
stressful sequelae include crowding, hypoxia, physical
trauma, aftereffects of anesthetics or sedatives, and baromet-
ric disturbance in sh harvested at considerable depth. Evi-
dence that these stimuli are intrinsically stressful is provided
by experiments that have documented marked increases in
blood cortisol and/or glucose levels in sh following deliber-
ate handling and transport (Acerete et al. 2004; Barton 2002;
Hosoya et al. 2007).
There may be some benet to sedating sh before trans-
port in order to mitigate shipping stress. In a study in which
channel catsh (Ictalurus punctatus) were subjected to stress-
ors such as connement, high ammonia, and oxygen deple-
tion, sedation resulted in lower cortisol elevations than those
observed in control sh (Small 2004). But the magnitude of
the stress response to netting, transport, and handling varies
considerably among species and, typical of stress responses
in sh, clinical effects often do not become apparent until
several days after the stress-inducing event, when secondary
bacterial, viral, fungal, or parasitic infections manifest.
Notwithstanding the frequency at which sh experience
these stressors, there has been very little investigation of po-
tential histomorphologic consequences. For example, although
anecdotal observations suggest that sh may suffer micro-
scopically evident muscle degeneration (rhabdomyolysis) as a
consequence of collection (capture myopathy), experiments
have not been conducted to conrm this causal relationship.
Crowding
For captive sh, appropriate stocking density varies greatly
according to the species, housing system, and available re-
sources. Overcrowding may be accompanied by additional
stressors such as poor water quality, exposure to organic
wastes, and conspecic aggression and predation. Gilthead
seabream (Sparus aurata L.) experienced signicant rapid
increases in blood cortisol and glucose following short-term
crowding (Ortuo et al. 2001), and similar results were ob-
served in tilapia (Oreochromis mossambicus) (Vijayan et al.
1997), thus supporting the role of crowding as a stressor. In
tilapia, glucose elevations after 2 hours of connement were
attributed to glycogenolysis, whereas in sh conned for 24
hours gluconeogenesis was considered the primary mecha-
nism for glucose elevations (Vijayan et al. 1997). Although
morphologic changes were not the focus of these experi-
ments, it is reasonable to surmise that certain durations of
connement stress might therefore manifest in histopatho-
logic ndings such as decreased hepatocellular vacuolation
(especially in cultured sh) and muscle atrophy. Burgess and
Coss (1982) examined histologic specimens from adult jewel
sh (Hemichromis bimaculatus Gill) and determined that
moderate crowding stress was associated with morphologic
changes in the brain.
Hyper- or Hypothermia
Fish are subject to stress from either rapid temperature uc-
tuations that preclude acclimation or inappropriate water
temperature (beyond the high or low range of tolerance). A
rapid temperature decrease limits a shs ability to produce
antibodies integral to an immediate immune response, and a
delay in the immune response may enable pathogens to colo-
nize, reproduce, and establish an infection. Very cold tem-
peratures may inactivate defensive functions of nonspecic
leukocytes known as natural killer (NK) cells, although there
is some evidence from studies in common carp (Cyprinus
carpio) that NK cells may be able to accommodate tempera-
ture changes over time (Kurata et al. 1995). Hyperthermia
has been used experimentally as a stressor in challenge stud-
ies involving infectious agents, for example in rainbow trout
(Oncorhynchus mykiss) exposed to Saprolegnia parasitica
(Gieseker et al. 2006). This same stressor also contributed to
altered thyroid indices, including augmentation of thyroid
epithelial cell height, in rainbow trout exposed to PCBs
(Buckman et al. 2007).
Hypoxia
Anoxic conditions are commonly the result of plant, algae,
or diatom overgrowth in either natural or captive environ-
ments, but hypoxia can also occur when sh are shipped in
insufciently aerated containers, for example. The decrease
in oxygen availability to tissues can lead to necrotic or apop-
totic lesions in organs (Geng 2003; van der Meer et al. 2005).
In channel catsh, experimentally induced sublethal hypoxia
was responsible for histopathologically evident necrosis, hy-
peremia (vascular congestion), edema, hemorrhage, hyper-
plasia, and/or hypertrophy in a variety of anatomic sites
including the gills, liver, spleen, and anterior and posterior
kidney (Scott and Rogers 1980). Although it could be rea-
sonably argued that such lesions formed as a specic reac-
tion to acute localized oxygen deprivation rather than to
stress per se, it is plausible that stress contributed to the re-
sponse on some level.
Some teleost sh, frogs, turtles, snakes, and insects have
the capacity to tolerate or adapt to hypoxia (van der Meer
et al. 2005). For instance, zebrash (Danio rerio) can survive
weeks of severe hypoxia through adaptive responses that
modulate their behavioral and physical phenotype: evidence
from cDNA microarray technology revealed changes in gene
expression in their gills as well as gene repression that affected
390 ILAR Journal
protein biosynthesis and metabolic pathways (van der Meer
et al. 2005).
More typically, however, chronic hypoxia has been shown
to cause an assortment of phenotypic changes in a diverse
range of organ systems and sh species, including the hearts
of zebrash and cichlids (Haplochromis piceatus) (Marques
et al. 2008); the reproductive tracts of common carp (Wang
et al. 2008) and Atlantic croaker (Micropogonias undulatus)
(Thomas et al. 2007); peripheral blood leukocytes of tilapia
(Choi et al. 2007); and the eyes of platysh (Xiphophorus
maculatus) exposed to hypoxic conditions perinatally (Chan
et al. 2007). In the gills, hypoxia has been associated with an
adaptive increase in lamellar surface area in shes such
as certain African cichlids and Crucian carp (Carassius
carassius) (Chapman et al. 2000; Sollid et al. 2003; van der
Meer et al. 2005).
Hyper- or Hyposalinity
Freshwater sh are under continuous pressure to conserve
salts, whereas the reverse is true for marine species, which
must conserve water (Greenwell et al. 2003). Among shes
in general, the ability to adapt to alterations in salinity varies
markedly and often is indirectly proportional to the pace of
the changes. In natural settings, salinity levels can uctuate
with tides, season, or evaporation from surface waters.
Few studies have investigated potential morphologic
effects of salinity as the sole stressor. An experiment to as-
sess optimal stocking densities for sea bass (Dicentrarchus
labrax) ngerlings applied hypersalinity as a stressor along
with temperature modications (Via et al. 1998). But an ex-
periment that specically evaluated the tolerance of hybrid
tilapia (Oreochromis mossambicus O. urolepis hornorum)
to hypersaline water found that the primary morphologic in-
dicators of hypersaline stress, and the most sensitive of sev-
eral endpoints tested, were ultrastructural changes in the
gills (Sardella et al. 2004).
In anadromous sh such as salmon, physiological changes
associated with smoltication (the metamorphic transforma-
tion that occurs in juveniles before their freshwater to marine
migration) are consistently stressful, as suggested by changes
in plasma cortisol levels (Barton 2002).
Malnutrition
Using a greatly simplied classication system, malnutrition
can be categorized as disorders that result from either (1) an
insufciency or overabundance of nutrients or (2) relative
nutrient imbalances. Factors that typically contribute to mal-
nutrition in wild sh include depletion of species-appropriate
food sources or components (e.g., vitamins, minerals),
height ened competition for available food resources, and
inappetence due to disease. Captive sh often endure the ad-
ditional challenge of suboptimal feed formulation, usually
because the precise nutritional requirements for the sh spe-
cies of interest have not been determined, a suitable diet can-
not be provided because of cost or lack of availability, or
nutrient degradation occurred during feed storage.
Because stress-reactive hormones such as glucocorticoids
have a constituent role in energy homeostasis, it is often dif-
cult to separate stress responses from the direct effects of
malnutrition in terms of morphologic consequences. For ex-
ample, starvation may cause a histologically evident decrease
in liver glycogen stores not only as a result of increased energy
expenditure relative to intake but also because of stress-
induced corticosteroid-mediated glycogenolysis (Barton and
Schreck 1987; Vijayan et al. 1997). Furthermore, food depri-
vation can lead to reduced stress resistance, as was the out-
come when food-denied Atlantic cod (Gadus morhua L.) were
subjected to exhaustive exercise (Olsen et al. 2008). Another
recent study further demonstrated that nutrient imbalances
can inuence the stress response, as higher blood cortisol con-
centrations in rainbow trout were associated with dietary vari-
ations of vitamin E, vitamin C, and highly unsaturated fatty
acids (Trenzado et al. 2008). In such cases it may be difcult
to discriminate the stressor from the stress response; for ex-
ample, interrenal ascorbic acid concentrations decreased in
rainbow trout and coho salmon (Oncorhynchus kisutch) that
were subjected to nonspecic stress (Wedemeyer 1969).
Contaminants
Fish have been exposed, either intentionally or unintention-
ally, to a vast array of chemical and particulate contaminants,
of both natural and man-made origin. Examples include
pharmaceuticals, agricultural chemicals, manufacturing by-
products, animal and human waste materials, mining efu-
ents, and substances released as a consequence of natural
disasters such as res. Arguably, at sufcient concentration,
almost any contaminant is capable of inducing a stress re-
sponse. In some exposures, the stressor is a mixture of known
and unknown contaminants (Dutta et al. 2005; Teh et al.
1997), in which case it is almost impossible to differentiate
stress response effects from manifestations of toxicity. How-
ever, such differentiation can be challenging even when the
contaminant is a single compound.
One of the most studied contaminants is ammonia, high
levels of which result from agricultural or mining operation
runoff, excessive biological waste accumulation, insufcient
water aeration, or inadequate tank conditioning (Noga 1996;
Randall and Tsui 2002; Spencer et al. 2008). Ammonia is
toxic to all vertebrates, and the effects of both acute and
chronic ammonia exposure have been investigated in a num-
ber of sh species. Acute ammonia toxicity can cause an as-
sortment of clinical signs in sh, the most severe of which
include convulsions, coma, and death (Randall and Tsui
2002), as well as less severe impacts such as plasma cortisol
elevations and behavioral changes such as hyperexcitability
and appetite suppression (Ortega et al. 2005). Its effects may
be exacerbated by increased pH or temperature, excessive
exercise, starvation, and stress (simulated by cortisol injec-
tion) (Randall and Tsui 2002; Spencer et al. 2008).
Volume 50, Number 4 2009 391
Ammonia exposure has been associated with morphologic
ndings in a variety of sh tissues. The gills are one of the
most frequently reported targets (Benli et al. 2008; Frances
et al. 2000; Lease et al. 2003; Spencer et al. 2008), although in
one study involving chronic ammonia toxicity in rainbow
trout, gill changes were not observed histologically, even in
high-dose sh that had suffered from neurological dysfunc-
tion (Daoust and Ferguson 1984). In addition to the gills,
ammonia-related lesions have been reported in the liver, kid-
ney, intestine, and ovary of sh (Banerjee and Bhattacharya
1994, 1995; Benli et al. 2008; Dey and Bhattacharya 1989).
Stress Responses
Fish responses to stress can be divided into three phases:
primary, secondary, and tertiary (Barton 2002). The primary
phase refers to a generalized neuroendocrine response in
which catecholamines (epinephrine and norepinephrine) and
cortisol are released from chromafn and interrenal cells,
respectively. Higher circulating levels of these hormones
trigger a secondary response that involves physiologic and
metabolic pathways; examples of the secondary response in-
clude hyperglycemia due to enhanced glycogenolysis and
gluconeogenesis, vasodilation of arteries in gill laments,
increased cardiac stroke volume, and immune function de-
pression (Gratzek and Reinert 1984). The rst two phases
are considered adaptive and enable sh to adjust to stressors
and maintain homeostasis. In contrast, tertiary responses in-
volve systemic changes in which animals may become inca-
pable of adapting to stressors, leading to adverse effects on
the animals overall health, including their performance,
growth, reproduction, disease resistance, and behavior (Barton
2002). The following sections provide examples of adaptive
and postadaptive stress responses according to organ system.
This is by no means an exhaustive record; undoubtedly, mor-
phologic indications of stress also exist in tissue types that
are less routinely examined.
Gills
Given the relative fragility of the gills compared to other sur-
face tissues, and the fact that they are continually exposed to
the shs external environment, it is remarkable that these
structures are able to survive and compensate for the chemi-
cal and physical assaults to which they are invariably sub-
jected. It is therefore not surprising that, based on a survey of
the literature, the gills appear to be a frequent target for stress
responses (Figure 1).
Some shes have developed intriguing adaptive stress
response mechanisms. For example, the gills of Crucian carp
exhibit a reversible morphological reaction to decreased ox-
ygen availability (Sollid et al. 2003), thanks to a unique ana-
tomic feature: under normal ambient oxygen concentrations,
the gills lack protruding secondary lamellae (typically the
primary sites of gas exchange in other shes); instead, the
secondary lamellae are embedded in a cell mass that, by de-
sign, decreases the respiratory surface area. Under hypoxic
conditions, this cell mass recedes due to the combined ef-
fects of increased apoptosis and diminished cell prolifera-
tion, and as it shrinks it exposes the underlying lamellae,
thus increasing the overall surface area of the gills. This ad-
aptation may have evolved to reduce water and ion ux
under normoxic conditions and thus conserve energy for
osmoregulation. Similarly, in various African cichlid sh ex-
posure to long-term hypoxia resulted in elongation of bran-
chial laments and an increase in the size of secondary
lamellae (Chapman et al. 2000).
Most species, however, are not capable of adapting so
effectively to hypoxic conditions. Channel catsh exposed
to varying degrees of sublethal hypoxia exhibited a suite of
nonspecic, histologically evident changes likely to inter-
fere with respiratory gas exchange, such as gill epithelial
hypertrophy and hyperplasia, goblet cell proliferation with
increased mucus secretion, hemorrhage, edema, and telangi-
ectasis (Scott and Rogers 1980).
Figure 1 Nonspecic stress response in the gills of adult Atlantic
salmon (Salmo salar L.). (A) Normal gill (two adjacent laments).
(B) Findings associated with several types of stressors; the most
prominent changes are mucus cell hyperplasia (arrow) and epithe-
lial lifting (arrowhead). Bar = 50 microns.
392 ILAR Journal
Hypersalinity results in a qualitatively different type of
negative response. Apoptosis of chloride cells (branchial cells
that facilitate ion transport and have an integral role in acid-
base regulation; Perry 1998) occurred in hybrid tilapia ex-
posed experimentally to various concentrations of hypersaline
water for a model of salinity tolerance (Sardella et al. 2004).
Ammonia-induced gill changes have been particularly
well characterized, for species as diverse as Nile tilapia
(Oreochromis nilotica), slimy sculpin (Cottus cognatus),
and endangered Lost River suckers (Deltistes luxatus). They
include nonspecic responses such as lamellar thickening,
mucus cell hyperplasia and hypertrophy, epithelial cell lift-
ing, leukocyte inltration, hyperemia, hemorrhage, chloride
cell proliferation, secondary lamellar fusion, and telangi-
ectasis (Benli et al. 2008; Lease et al. 2003; Spencer et al.
2008). After an investigation of the combined effects of am-
monia and elevated pH in Lost River suckers, Lease and col-
leagues (2003) concluded that structural gill changes were
more sensitive than other traditional assays for detecting am-
monia toxicity. An earlier study recorded similar types of
morphologic ndings in wild freshwater sh exposed to a
mixture of known and unknown contaminants (Teh et al.
1997). Gill lesions in that study included hyperplastic mu-
cous and chloride cells, deformed branchial cartilages, se-
vere and diffuse lamellar aneurysms (telangiectasis), and
edema at the bases of secondary lamellae.
At this point it may seem that any type of stressor might
induce almost any type of gill lesion as part of a stress re-
sponse. But apparently this is not necessarily the case, as one
study has demonstrated that social stress did not lead to chlo-
ride cell proliferation in rainbow trout (Sloman et al. 2005).
Liver
Unlike the gills, the liver is clearly protected from physical
exposure to the external environment, at least under normal
circumstances. It is prone, however, to chemical assault, in
part due to an efcient enterohepatic cycling mechanism
(Gingerich 1982). Stress responses may also be evident in
the liver because of its prominent role in energy storage and
metabolism. Often, quantitative alterations in hepatic energy
storage are visible macroscopically as changes in liver size
and coloration, and histologically as variations in hepatocel-
lular vacuolation and tinctorial staining characteristics (Wolf
and Wolfe 2005). Decreased vacuolation can result from loss
of cytoplasmic glycogen and/or lipid caused by insufcient
energy intake relative to need and/or glucocorticoid-induced
glycogenolysis. Conversely, increased hepatocellular vacu-
olation is more commonly associated with overnutrition or
toxicity (Wolf and Wolfe 2005). As an example of the latter,
cloudy swelling and hydropic degeneration occurred in Nile
tilapia exposed to sublethal concentrations of ammonia
(Benli et al. 2008). On the other hand, alterations in cyto-
plasmic vacuolation were not features of hypoxia in channel
catsh, which instead showed hepatic necrosis and hemor-
rhage as well as splenic changes such as edema, hyperemia,
and necrosis. Chronic histopathologic changes such as se-
vere hepatic lipidosis, lymphoid cell depletion, vascular con-
gestion, and reticuloendothelial cell necrosis (in the spleen)
were evident in the livers and spleens of wild freshwater sh
exposed to mixed contaminants (Teh et al. 1997). The added
presence of a number of preneoplastic and neoplastic prolif-
erative lesions in those sh strongly suggests that factors
other than glucocorticoid-mediated stress (e.g., chemical
carcinogenesis, patent toxicity) may have contributed to at
least some of the chronic changes.
Integument
The skin, with its scales and surface mucus, provides a protec-
tive physical barrier that is important in terms of both osmo-
regulation and pathogen defense. But sh skin is susceptible
to damage from handling, ghting, physical trauma, preda-
tion, environmental irritants, and pathogens, and the damage
can lead to opportunistic microbial infections. At that stage
the stress response may further compromise the hosts de-
fenses, via corticosteroid-mediated immunosuppression or
other stress-related immunosuppressive factors (Choi et al.
2007; Harris et al. 2000; Kent and Hedrick 1987).
Although sh skin has not been reported extensively as a
stress response target, dermal ulceration was the chief nding
in a series of studies in which striped bass (Morone saxatilis)
and striped bass hybrids were exposed to acute connement
stress (Noga et al. 1998; Udomkusonsri et al. 2004). Associ-
ated histopathologic lesions, in addition to rapidly occurring
epithelial erosions and ulcers that primarily affected the ns,
included epithelial cell swelling, edema of the dermis and
hypodermis, melanophore aggregation, and stromal tissue
necrosis.
Genitourinary Tract
Although there are reports of functional and/or hormonal
impairment of the sh reproductive system due to various
stressors (capture, handling, crowding, hypoxia, tank drain-
ing, noise) (Cleary et al. 2002; Contreras-Snchez et al.
1998; Thomas et al 2007; Wang et al. 2008), there has been
only limited investigation of the potential morphologic ef-
fects of such stressors in the gonads or genital ducts. One
study found retarded oocyte maturation in common carp ex-
posed to chronic hypoxia (Wang et al. 2008). In another
study conducted in Atlantic croaker, hypoxia was associated
with decreased gonadosomatic index (gonadal weight/body
weight) and impaired gametogenesis (determined via mor-
phometric counting of ovarian and testicular germ cells in
histologic sections) in both male and female sh (Thomas
et al. 2007).
There are even fewer reports of stress responses that
involve the sh urinary tract. Examples include hypoxia-
induced hemorrhage, glomerular congestion, and edema in
the posterior kidneys of channel catsh (Scott and Rogers
1980), and congestion in Nile tilapia exposed to sublethal
concentrations of ammonia (Benli et al. 2008).
Volume 50, Number 4 2009 393
Nervous and Sensory Systems
Routine diagnostic examinations or experimental investiga-
tions involving sh tissues tend to include sampling of the
brain and spinal cord less frequently than for other organs.
Although inammation and endoparasitism of the central
nervous system are often readily recognizable in standard
histologic sections, more subtle types of changes are not al-
ways easily appreciated. For example, in a series of experi-
ments in which jewel sh were exposed to chronic crowding
stress, special histologic staining and morphometric tech-
niques were required in order to determine that, compared to
controls, crowded sh had structural nerve cell alterations
(both qualitative and quantitative) in the optic tectum, a ma-
jor area of the brain concerned with processing and integrat-
ing sensory information (Burgess and Coss 1982). Of course
it could be debated that the outcome was not truly a stress
response but instead a developmental adaptation caused by
long-term differences in patterns of sensory stimulation.
Comparable to the central nervous system, the detec-
tion of stress-related changes in the eyes may also require
detailed examination. For example, ndings in perinatal
platysh subjected to hypoxic conditions included central
corneal thinning, hyperplasia of corneal endothelial cells,
lens ber derangement, and apoptotic cells in the retina
(Chan et al. 2007). Perhaps more obvious were the corneal
ulcerations induced by acute connement stress in hybrid
striped bass (Morone saxatilis M. chrysops) (Udomkusonsri
et al. 2004).
Cardiovascular System
Histologically evident changes in the hearts of adult ze-
brash and Lake Victoria cichlids (Haplochromis piceatus)
subjected to chronic hypoxia included reduced ventricular
outow tracts and reduced lacunae surrounding trabeculae
(Marques et al. 2008). Quantitation of myocyte nuclei in
both species also revealed that, relative to controls, hypoxic
sh had increased numbers of nuclei per unit area.
Occasionally, microscopic examinations of blood smears
can reveal morphologic evidence of stress that would be dif-
cult to detect in tissue sections. For example, two classic
hematological manifestations of the stress response in mam-
mals, neutrophilia and lymphopenia, were triggered in Nile
tilapia by acute hypoxia followed by reperfusion (Choi et al.
2007). Although a description of hematological changes as-
sociated with stress is outside the scope of this article, many
publications clearly indicate that stressors such as handling,
crowding, capture, restraint, hypoxia, anesthesia, air expo-
sure, and sampling technique can affect sh hematology
and/or clinical chemistry values (Dror et al. 2006; Ellsaesser
and Clem 1987; Fast et al. 2007; Gbore et al. 2006; Greenwell
et al. 2003; Groff and Zinkl 1999; Scott and Ellis 2007). In
addition, evaluation of myoglobin seems to be relevant in the
evaluation of hypoxic stress in sh; for example, recent evi-
dence indicates that unique types of myoglobin are present
in many different sh tissues including blood vessels in hy-
poxia-tolerant sh (Cossins et al. 2009).
Multiorgan and Systemic Stress Responses
Systemic stress responses include alterations (often de-
creases) in body condition and/or organ weights, with cor-
responding histopathologic changes such as atrophy of
adipose tissue (fat), skeletal and cardiac muscle, and liver
cells, among other tissue types (Figure 2).
One particular multiorgan stress response involves the for-
mation of histologically evident pigmented macrophage ag-
gregates (PMA; Figure 3). These melanomacrophage centers
are variably sized constituent nests of phagocytic cells that can
contain one or more intracytoplasmic pigments, such as ceroid,
lipofuscin, melanin, and hemosiderin (Wolke 1992). Although
the kidney and spleen tend to be common locations for these
Figure 2 Histomorphologic effects of chronic starvation in adult
female Japanese medaka (Oryzias latipes). Images (A), (C), and (E)
are from a well-nourished sh; (B), (D), and (F) are from a sh that
suffered a prolonged negative energy balance due to inanition and
stress associated with egg retention. (A) Normal skeletal muscle.
(B) Skeletal muscle atrophy; muscle cell nuclei (arrows) appear
clumped as a result of the decrease in muscle ber size. (C) Normal
liver; arrowheads indicate moderate hepatocyte vacuolation consis-
tent with glycogen storage. (D) Liver atrophy; the tissue is barely
recognizable as liver because hepatocytes are severely shrunken
and there is a loss of vacuolation due to glycogen depletion. (E)
Normal kidney; epithelial cells of a renal tubule (arrow) have abun-
dant eosinophilic (pink) cytoplasm, and hematopoietic tissue (H) is
plentiful. (F) Kidney atrophy; arrow indicates a shrunken tubule.
(A, B): bar = 100 microns; (CF): bar = 250 microns.
394 ILAR Journal
aggregates, PMA may also be found in the liver, heart, gonads,
and many other anatomic sites. The predilection for PMA to
be present in certain tissues rather than others, and the pigment
constitution of PMA, both tend to be species dependent
(Schwindt et al. 2006). Whenever possible, PMA should be
differentiated from foci of granulomatous inammation, which
are more typically a response to microbial infection, for ex-
ample. Some of the many functions attributed to PMA include
sequestration of cell breakdown products, recycling and stor-
age of iron, antigen presentation, and detoxication of exoge-
nous and endogenous substances (Agius and Roberts 1981;
Ellis 1980; Herraez and Zapata 1986; Mori 1980). PMA tend
to increase in number and/or size as sh age, but reports indi-
cate that proliferation of these structures may also occur as a
nonspecic response to various stressors, such as heat (Blazer
et al. 1987), starvation (Agius and Roberts 1981; Herraez and
Zapata 1986), and nutritional imbalance (Moccia et al. 1984).
The potential importance of PMA as a tool for monitoring
stress is evident in recent efforts to quantify these aggregates
morphometrically in histologic sections (Jordanova et al.
2008; Russo et al. 2007; Schwindt et al. 2006).
Conclusions
For several reasons, scientists understanding of stress re-
mains nebulous. First, the interplay between stressors and
stress responses is highly complex, and some stress responses
may themselves function as stressors, and vice versa. Sec-
ond, there are few, if any, pathognomonic stress responses
(the nonspecic nature of stress responses is in keeping with
Selyes original denition). A third explanation concerns the
tendency of researchers to use the term stress to indicate
almost any type of adverse condition that a sh might en-
counter or any form of outcome. Purists may argue, some-
what justiably, that at least some of the stress responses
discussed in this review are not actually the result of stress
per se because they are not necessarily mediated by stress
hormones. Thus, exposure to pollutants may indeed be stress-
ful, but the associated morphologic effects may actually re-
ect tissue damage due to toxic mechanisms or specialized
physiologic adaptations to an unfavorable environment.
Notwithstanding these reasons for lack of clarity, in live
animal research it is important to recognize the potential for
stress, however dened, to confound a studys results. Fail-
ure to do so is likely to lead to erroneous conclusions that
may be perpetuated in the literature. Moreover, scientists
must determine the extent to which certain effects are attrib-
utable to a particular stressor under specied conditions.
Further challenge studies of sh may enhance understanding
of stress and its effects in sh through the administration of
glucocorticoid or adrenergic hormones, heat shock proteins,
or other types of mediators not yet identied.
Acknowledgments
Funding for this project was provided in part by Experimen-
tal Pathology Laboratories Inc., in Sterling, Virginia.
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