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Morphologic Effects of the Stress Response in Fish

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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 immediate surroundings. Yet few studies have addressed stress as a potential confounding factor for bioassays that use fish as test subjects. Common stressors encountered by captive fish 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 fish: (1) stress is an amorphous term that does not have a consistently applied definition; (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 difficult to distinguish from tissue damage or compensatory adaptations induced specifically by the stressor. Further investigations are necessary to more precisely define the role of stress in the interpretation of fish research results.
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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 fi sh as
test subjects. Common stressors encountered by captive fi 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 fi sh: (1) stress is an
amorphous term that does not have a consistently applied
defi nition; (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 diffi cult to dis-
tinguish from tissue damage or compensatory adaptations
induced specifi cally by the stressor. Further investigations
are necessary to more precisely defi ne the role of stress in
the interpretation of fi sh research results.
Key Words: contaminant; crowding; fi sh; handling; histol-
ogy; nutrition; temperature; salinity; stress
Introduction
In 1936 a scientist named Hans Selye, upon observing
effects of noxious stimuli in laboratory animals, coined
the term “stress” and defi ned it as “the non-specifi c 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 defi nitions, 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 individual’s physical or
mental awareness of that stimulus, or (3) the individual’s
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 fi sh as determined by gross or
histopathologic investigation. In addition, descriptions of
both tissue-specifi c and non-tissue-specifi c 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), insuffi cient 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, fi 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 fi sh include fl uctuations
in water salinity, pH, hardness, alkalinity, dissolved solids,
water level or current, and exposure to waterborne pathogens
or toxicants. Fish reared in confi nement 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 fi ght or fl 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 fi sh lack adrenal glands per se,
analogous production and release of adrenal cortical and
medullary hormones occur in the interrenal cells and chro-
maffi n 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 fi sh can be a double-edged
sword. The activities of these hormones are clearly benefi -
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 infl ammatory 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 fi 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 fi 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 diffi cult 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 fi 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 fi 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 fi 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 specifi cally 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-Sánchez 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 fi sh gonads or gonadal ducts; such
effects might include increased germ cell degeneration in the
ovary (oocyte atresia) and/or testis. Such fi ndings would be
signifi cant 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 fi sh gonads and lead
to morphological changes (Abe and Munehara 2007).
Fish-Specifi c Stressors
There are roughly 30,000 known species of fi sh, and both
wild and captive fi 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 fi 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
Specifi c histopathologic fi 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 fi sh, but wild fi sh may also experience these distur-
bances, for example through catch and release programs in
recreational fi sheries. Procedures that can intensify the stress
response in aquacultured fi 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 fi 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 fi sh following deliber-
ate handling and transport (Acerete et al. 2004; Barton 2002;
Hosoya et al. 2007).
There may be some benefi t to sedating fi sh before trans-
port in order to mitigate shipping stress. In a study in which
channel catfi sh (Ictalurus punctatus) were subjected to stress-
ors such as confi nement, high ammonia, and oxygen deple-
tion, sedation resulted in lower cortisol elevations than those
observed in control fi 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 fi 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 fi sh experience
these stressors, there has been very little investigation of po-
tential histomorphologic consequences. For example, although
anecdotal observations suggest that fi sh may suffer micro-
scopically evident muscle degeneration (rhabdomyolysis) as a
consequence of collection (capture myopathy), experiments
have not been conducted to confi rm this causal relationship.
Crowding
For captive fi 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 conspecifi c aggression and predation. Gilthead
seabream (Sparus aurata L.) experienced signifi cant rapid
increases in blood cortisol and glucose following short-term
crowding (Ortuño 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 confi nement were
attributed to glycogenolysis, whereas in fi sh confi ned 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
confi nement stress might therefore manifest in histopatho-
logic fi ndings such as decreased hepatocellular vacuolation
(especially in cultured fi 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 fl uc-
tuations that preclude acclimation or inappropriate water
temperature (beyond the high or low range of tolerance). A
rapid temperature decrease limits a fi sh’s 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 nonspecifi c
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 fi sh are shipped in
insuffi ciently 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 catfi sh, 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 specifi c 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 fi sh, frogs, turtles, snakes, and insects have
the capacity to tolerate or adapt to hypoxia (van der Meer
et al. 2005). For instance, zebrafi sh (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 fi sh species, including the hearts
of zebrafi sh 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 platyfi sh (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 fi 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 fi 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 fi 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 fl 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) fi ngerlings applied hypersalinity as a stressor along
with temperature modifi cations (Via et al. 1998). But an ex-
periment that specifi cally 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 fi sh such as salmon, physiological changes
associated with smoltifi cation (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 simplifi ed classifi cation system, malnutrition
can be categorized as disorders that result from either (1) an
insuffi ciency or overabundance of nutrients or (2) relative
nutrient imbalances. Factors that typically contribute to mal-
nutrition in wild fi 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 fi sh often endure the ad-
ditional challenge of suboptimal feed formulation, usually
because the precise nutritional requirements for the fi 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 infl uence 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 diffi cult
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 nonspecifi c 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 effl u-
ents, and substances released as a consequence of natural
disasters such as fi res. Arguably, at suffi cient 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, insuffi cient
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 fi sh species. Acute ammonia toxicity can cause an as-
sortment of clinical signs in fi 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 fi 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 fi 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 fi 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 chromaffi n 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 fi laments,
increased cardiac stroke volume, and immune function de-
pression (Gratzek and Reinert 1984). The fi rst two phases
are considered adaptive and enable fi 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 fi sh’s 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 fi 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 fi 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 fl ux
under normoxic conditions and thus conserve energy for
osmoregulation. Similarly, in various African cichlid fi sh ex-
posure to long-term hypoxia resulted in elongation of bran-
chial fi 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 catfi sh exposed
to varying degrees of sublethal hypoxia exhibited a suite of
nonspecifi c, 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 Nonspecifi c stress response in the gills of adult Atlantic
salmon (Salmo salar L.). (A) Normal gill (two adjacent fi 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 nonspecifi c responses such as lamellar thickening,
mucus cell hyperplasia and hypertrophy, epithelial cell lift-
ing, leukocyte infi ltration, 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 fi ndings in wild freshwater fi 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 effi cient 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 insuffi cient
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
catfi sh, 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 fi sh
exposed to mixed contaminants (Teh et al. 1997). The added
presence of a number of preneoplastic and neoplastic prolif-
erative lesions in those fi 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 fi sh skin is susceptible
to damage from handling, fi 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 host’s 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 fi sh skin has not been reported extensively as a
stress response target, dermal ulceration was the chief fi nding
in a series of studies in which striped bass (Morone saxatilis)
and striped bass hybrids were exposed to acute confi nement
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 fi 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 fi sh reproductive system due to various
stressors (capture, handling, crowding, hypoxia, tank drain-
ing, noise) (Cleary et al. 2002; Contreras-Sánchez 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 fi sh (Thomas
et al. 2007).
There are even fewer reports of stress responses that
involve the fi sh urinary tract. Examples include hypoxia-
induced hemorrhage, glomerular congestion, and edema in
the posterior kidneys of channel catfi sh (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 fi sh tissues tend to include sampling of the
brain and spinal cord less frequently than for other organs.
Although infl ammation 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 fi 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 fi 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, fi ndings in perinatal
platyfi sh subjected to hypoxic conditions included central
corneal thinning, hyperplasia of corneal endothelial cells,
lens fi ber derangement, and apoptotic cells in the retina
(Chan et al. 2007). Perhaps more obvious were the corneal
ulcerations induced by acute confi nement stress in hybrid
striped bass (Morone saxatilis × M. chrysops) (Udomkusonsri
et al. 2004).
Cardiovascular System
Histologically evident changes in the hearts of adult ze-
brafi sh and Lake Victoria cichlids (Haplochromis piceatus)
subjected to chronic hypoxia included reduced ventricular
outfl ow 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 fi 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 fi sh; for example, recent evi-
dence indicates that unique types of myoglobin are present
in many different fi sh tissues including blood vessels in hy-
poxia-tolerant fi 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 fi sh; (B), (D), and (F) are from a fi 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 fi 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; (C–F): 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 infl ammation, 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 detoxifi cation 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 fi sh age, but reports indi-
cate that proliferation of these structures may also occur as a
nonspecifi c 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 nonspecifi c nature of stress responses is in keeping with
Selye’s original defi nition). A third explanation concerns the
tendency of researchers to use the term “stress” to indicate
almost any type of adverse condition that a fi sh might en-
counter or any form of outcome. Purists may argue, some-
what justifi ably, 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 defi ned, to confound a study’s 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 specifi ed conditions.
Further challenge studies of fi sh may enhance understanding
of stress and its effects in fi sh through the administration of
glucocorticoid or adrenergic hormones, heat shock proteins,
or other types of mediators not yet identifi ed.
Acknowledgments
Funding for this project was provided in part by Experimen-
tal Pathology Laboratories Inc., in Sterling, Virginia.
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... A positive correlation between hepatocyte vacuolisation and PFAS accumulation in the liver was shown also in eels exposed to PFOA [32], zebrafish exposed to PFOS [31], and rats exposed to PFOA and PFOS [33]. The increase in vacuolation could be considered a defence mechanism against the toxic effects of PFAS. ...
... This study also shows that vacuolisation is species-dependent and, in the case of PFAS pollution, might correlate with the habits of the species. Decreased vacuolisation can result from a reduction in the cytoplasmic glycogen and/or lipids caused by insufficient energy intake or the use of resources due to glucocorticoid-induced glycogenolysis [33]. It is, therefore, presumable to hypothesise that this species, notoriously potamodromous, responded to xenobiotic stress through greater use of energy resources. ...
... This phenomenon is commonly associated with overnutrition or chronic toxicity. It has already been observed in other studies, for example, in zebrafish fry chronically exposed to PFOS [33,38]. ...
Article
Full-text available
In recent decades, the interest in PFAS has grown exponentially around the world, due to the toxic effects induced by these chemical compounds in humans, as well as in other animals and plants. However, current knowledge related to the antistress responses that organisms can express when exposed to these substances is still insufficient and, therefore, requires further investigation. The present study focuses on antioxidant responses in Squalius cephalus and Padogobius bonelli, exposed to significant levels of PFAS in an area of the Veneto Region subjected to a recent relevant pollution case. These two ubiquitous freshwater species were sampled in three rivers characterised by different concentrations of PFAS. Several biomarkers of oxidative stress were evaluated, and the results suggest that PFAS chronic exposure induces some physiological responses in the target species, at both cellular and tissue scales. The risk of oxidative stress seems to be kept under control by the antioxidant system by means of gene activation at the mitochondrial level. Moreover, the histological analysis suggests an interesting protective mechanism against damage to the protein component based on lipid vacuolisation.
... A positive correlation between hepatocyte vacuolisation and PFAS accumulation in the liver was shown also in eels exposed to PFOA [32], zebrafish exposed to PFOS [31], and rats exposed to PFOA and PFOS [33]. The increase in vacuolation could be considered a defence mechanism against the toxic effects of PFAS. ...
... This study also shows that vacuolisation is species-dependent and, in the case of PFAS pollution, might correlate with the habits of the species. Decreased vacuolisation can result from a reduction in the cytoplasmic glycogen and/or lipids caused by insufficient energy intake or the use of resources due to glucocorticoid-induced glycogenolysis [33]. It is, therefore, presumable to hypothesise that this species, notoriously potamodromous, responded to xenobiotic stress through greater use of energy resources. ...
... This phenomenon is commonly associated with overnutrition or chronic toxicity. It has already been observed in other studies, for example, in zebrafish fry chronically exposed to PFOS [33,38]. ...
Preprint
In recent decades, the interest in PFAS has grown exponentially around the world, due to the toxic effects induced by these chemical compounds in humans, as well as in other animals and in plants. However, current knowledge related to the antistress responses that organisms can express when exposed to these substances is still insufficient and therefore it requires further investigation. The present study focuses on antioxidant responses in Squalius cephalus and Padogobius bonelli, exposed to significant levels of PFAS in an area of the Veneto region subjected to a recent relevant pollution case. These two ubiquitous freshwater species were sampled in three rivers characterized by different concentrations of PFAS. Several biomarkers of oxidative stress have been evaluated and the results suggest that PFAS chronic exposure induces some physiological responses in the target species, at both cellular and tissue scales. The risk of oxidative stress seems to be kept under control by the antioxidant system by means of gene activation at the mitochondrial level. Moreover, the histological analysis suggests an interesting protective mechanism against damage to the protein component based on lipid vacuolization.
... Especially at a low water supply and -exchange, pathogens can accumulate in fish tanks. Transcript levels indicative of bacterial or parasitic infection such as NOS2 and TLR5, TLR20 and TLR22 were highest in individuals on Farm 1. High HSI levels as well can be indicative of a potential stress response, with the liver as dominant metabolic organ (Harper and Wolf, 2009). GPX indicating oxidative stress (Meiler et al., 2020) was downregulated in individuals from Farm 1. Cataracts found especially on Farm 1 can be an indication for repeated stress, osmotic imbalance, oxidative stress on the lens fiber, rapid growth (Bjerkås and Sveier, 2004), nutrient deficiency (Breck et al., 2003(Breck et al., , 2005Waagbø et al., 2003Waagbø et al., , 2010Bjerkås and Sveier, 2004), water temperature fluctuations (Bjerkås et al., 2001;Remø et al., 2011) or the influence of carbon dioxide (Neves and Brown, 2015). ...
... Notably, trout on Farm 5 had the highest kidney scores. The kidney secretes hormones stimulating stress response pathways (Harper and Wolf, 2009). Genes with important roles in inflammatory/immune (HMOX1, IKBA_3, TNIP2, STAT1, IRF1, CEBPB, IFNG_2, CCL4, CC-chemokines, SOCS1 and PRF) and stress responses (UCP2) were highly upregulated on Farm 5 compared to Farm 4, which could be indicative for inflammation or potentially unknown suboptimal environmental and/or management conditions. ...
Article
The species-appropriate treatment of fish is of great importance and requires the accurate assessment of the fish welfare. Aim of this study was to get a deeper insight into the welfare of rainbow trout (Oncorhynchus mykiss) during grow-out on six farms equipped with flow through systems, each providing different environmental (water supply, and -exchange, water quality) and management conditions (stocking density, feeding frequency). The mRNA levels of 91 genes involved in stress and immune responses were analysed using multiplex quantitative PCR and complemented with organ histopathology, blood glucose and lactate levels, organ somatic indices, external morphological damage, behaviour, as well as environmental and management conditions. The farms analysed revealed large differences in environmental and management parameters. Accordingly the expression profiles of distinct sets of genes and other welfare indicators differed significantly between farms. Generally, fish from farms with a high water supply and fast water exchange at lower stocking densities had lower levels of external morphological damage. Stocking density and the duration of a total water exchange in the tank revealed a significant impact on gene expression. Further, effects of water quality and immunological responses were observed. The gene expression profiles obtained provide a deeper insight on the stress and immune response influenced by different rearing conditions, and hence a more accurate status of trout welfare in aquaculture settings.
... Fish gill is in direct contact with the external aquatic environment and plays an important role in coping with hypoxia. Hypoxic stress affects normal fish growth (Nilsson 2007) and causes pathological changes such as swelling, hypertrophy, hyperplasia, and tissue necrosis of fish gills (Sollid et al. 2005;Harper and Wolf 2009;Victori et al. 2011). The large numbers of blood vessels and capillaries in the gill filaments of scaleless carp provide a histological basis for their adaptation to low-oxygen environments (Qin et al. 2010). ...
Article
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Scaleless carp (Gymnocypris przewalskii) are well adapted to low oxygen environment, but their specific adaptation mechanism to hypoxic condition remains unclear. The gill is an important respiratory organ that plays a crucial role in regulating hypoxic stress. Here, we established fish hypoxic stress model, as well as investigated oxidative stress, apoptotic responses, and relative enzyme activities in the gills of scaleless carp after exposure to various levels of hypoxic stress. The results demonstrated that gill lamellar height and basal length increased significantly under severe hypoxic stress, and interval lengths between lamellae increased significantly under hypoxic stress. Furthermore, lamellar epithelial cells underwent apoptosis, cytoplasmic contraction, and mitochondrial expansion, and the number of apoptotic cells increased significantly after exposure to severe hypoxic stress for 24 h. Subsequently, Bcl-2 and Caspase 3 mRNA levels, as well as Bcl-2/Bax expression ratio were significantly increased after exposure to severe hypoxic stress for 24 h, indicating upregulation of anti-apoptotic processes. Moreover, malondialdehyde and hydrogen peroxide levels were significantly increased after exposure to hypoxic stress for 24 h. Superoxide dismutase activity increased significantly after exposure to severe hypoxia for 8 h and then decreased, while glutathione peroxidase activity and total antioxidant capacity increased significantly under hypoxic stress. Taken together, the results indicated that scaleless carp gills respond to acute hypoxic conditions by undergoing lamellar morphology remodeling, enhanced apoptosis, and increased antioxidant enzymatic activity. The study findings provided new insight into the adaptation mechanisms of scaleless carp in response to hypoxic challenge.
... Wild populations naturally experience a variety of adverse conditions, from attack by predators, starvation, or exposure to poor environmental conditions, including global warming, pollution, emerging pathogens, and overfishing [1]. However, farmed species are additionally exposed to stressful conditions caused by husbandry and management procedures related to high stocking density, suboptimal water quality, nutritional intensity and imbalanced diet, disease treatments, and inability to choose the most favorable living conditions [2][3][4]. Fish respond to adverse conditions with a series of changes at molecular, physiological, and whole-organism levels. Therefore, analysis of biochemical status, including leukocyte profile, gene expression of stress proteins, and histological tissue analysis are important tools providing information about the levels of stress, metabolic, functional, and morphological disorders of tissues reflecting a whole-organism function [5,6]. ...
Article
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The aim of this study was to compare the organismal responses of farmed and wild-caught turbot, Scophthalmus maximus, based on analyses of biochemical plasma parameters, leukocyte profile, and histological tissue profile of gills, kidney, liver, intestine, and spleen, as well as gene expression of stress proteins in kidney and liver tissue. The results revealed significant differences in plasma triglycerides (TRIG), total protein (TP), albumin (ALB), globulin (GLOB), bilirubin (TBIL), creatinine (CRE) levels, creatine kinase (CK), and superoxide dismutase (SOD) activities that were higher, and A/G ratio, calcium (Ca) and phosphorus (P) concentrations, alkaline phosphatase (ALP), and glutathione peroxidase (GPx) activity, which were lower in farmed population. The neutrophil-leukocyte (N:L) ratio and gene expression of HSP70, HSP90, and WAP65-2 were increased in the wild-caught turbot. The wild-caught turbot were infested with the gill digenean parasite Dactylogyrus sp. and tapeworm Bothriocephalus scorpii. The obtained results provide valuable data for the assessment of the physiological responses of turbot for future comparative studies of the effects of various endogenous and exogenous factors on homeostasis of this species.
Article
We investigated the effects of different concentrations of longevity spinach, Gynura procumbens, on the hematological parameters of acutely stressed Nile tilapia, Oreochromis niloticus, (average weight 461.81 ± 16.60 g and average length 28.71 ± 0.34 cm) and determined the best concentration. The fish were subjected to hormonal stress in this research. We fed the stress control group commercial feed with 0.01% hydrocortisone, a stress hormone (0.01% of fish body weight) without Gynura. All the treatment groups were supplemented with Gynura extracts (0.5 g/kg, 1.0 g/kg, and 1.5 g/kg of feed weight) in combination with hydrocortisone. We evaluated blood glucose, lysozyme activity, phagocytic capacity, hematocrit, spleen somatic index, and hepatosomatic index. During the acute stress period, G. procumbens has been shown to decrease the levels of blood glucose in 1.5 g/kg treatment group (49.60 mg/dl at Day 1; 53.75 mg/dl at Day 3) compared to stress control group (80.00 mg/dl at Day 1; 69.20 mg/dl at Day 3). Higher lysozyme activity observed in 1.5 g/kg Gynura treatment group (11.44 T/min at 540 nm) compared to control (7.85 T/min at 540 nm). The 1.5 g/kg treatment group maintained the homeostatic level of significant physiological parameters including phagocytic capacity, packed cell volume, and hepatosomatic index. These findings are promising for the development of new nutraceuticals for the aquaculture industry.
Article
The objective of this study was to investigate the effects of air replenishers on the growth, body composition, energy budget, and body morphology of four fish species through simulation within a fully submersible cage environment. The experimental species included Paralichthys olivaceous, which experiences swim bladder degeneration, and Lateolabrax japonicus, Oncorhynchus mykiss, and Cyprinus carpio, which are physoclistous, physostomous, and physostomous, respectively, with each species (N = 5) having an initial weight of 69.12 g, 73.10 g, 55.57 g, and 74.65 g, respectively. The four species were investigated in each trial designed with three different equipment for a 35-day period. The fish in the control group (Ctrl) were cultured in the tank with airstones, the mesh group (ME) used a mesh to keep the experimental fish from the water surface and airstones above the mesh, and the replenisher group (RM) had an air replenisher and airstones under the mesh. The results showed that there were no significant differences in the growth, body composition, energy budget, and body morphology of L. japonicus and P. olivaceus in different trials. For O. mykiss and C. carpio, compared to the Ctrl and RM groups, the specific growth ratio (reduced by 28.2% and 42.7%, respectively), protein content (reduced by 2.5% and 16.8%, respectively), and fat content (reduced by 14.2% and 27.2%, respectively) decreased. The proportion of feeding energy allocated to growth in the ME group also decreased, whereas that lost in respiration increased. An air replenisher can reduce the impact on the isolation of O. mykiss and C. carpio from the water's surface. Additionally, the streamlining of the body of O. mykiss in the ME group significantly decreased, whereas C. carpio in the ME group became more slender. This study implies that L. japonicus and P. olivaceus are not affected by isolation from the surface in a 1-month submergence culture, such as in a submersible cage, and the same effects on O. mykiss and C. carpio can be mitigated by air replenishment. Moreover, O. mykiss and C. carpio may adapt to negative buoyancy in submergence by changing body shape which generates more lift during swimming.
Article
The study evaluated the effect of different potassium supplementation dosages on the physiological responses of Pangasianodon hypophthalmus reared in an aquaponic system with Spinacia oleracea L. for 60 days. The system comprised of a rectangular fish tank of 168 l capacity (water volume=100 l) with Nutrient Film Technique (NFT) based hydroponic component with fish to plant ratio of 2.8 kg m‐3: 28 plants m‐2 in all the treatments. The osmoregulatory and stress parameters of P. hypophthalmus at four different potassium dosages of T1 (90 mg l‐1), T2 (120 mg l‐1), T3 (150 mg l‐1), and T4 (180 mg l‐1) were compared with C (control, 0 mg l‐1) to examine the potassium level to be applied to aquaponics. The water quality parameters and fish production were found to have no adverse impact due to potassium supplementation. The spinach yield during two harvests, i.e., before and after potassium supplementation, revealed that the yield was significantly higher (p<0.05) after supplementation with the highest yield in T3 and T4. The osmoregulatory parameters such as plasma osmolality, Na+, K+ ATPase activity in gill and plasma ionic profile (Cl‐, Ca2+ and Na+) showed an insignificant variation (p>0.05) between control and treatments except for higher plasma potassium concentration (1.98±0.19 mmol l‐1) in T4. The stress and antioxidant enzymes analysis exhibited significantly higher plasma glucose and SOD activity in gill and liver in T4, while cortisol and catalase showed an insignificant difference (p>0.05). The experimental findings demonstrated that the potassium dosage up to 150 mg l‐1 could be suggested as optimum for P. hypophthalmus and spinach aquaponics without impairing the health and oxidative status of P. hypophthalmus. This article is protected by copyright. All rights reserved.
Article
Hypoxia is thought to suppress the immune response and lead to severe mortality of marine organisms. In recent years, hypoxic stress has become a serious problem in Takifugu rubripes culture due to the increase in culture density. Gills, as the primary organ of physiological exchange with the surrounding environment, play a key role in the response of fish facing hypoxic stress. To understand the biological processes and complex molecular mechanisms of hypoxia in T. rubripes, we performed a transcriptomic analysis of hypoxic stress in the gills of T. rubripes. Twenty-four healthy fish weighing 461.75 ± 40.66 g were randomly divided into four groups according to oxygen concentration. [a control group (ppm5.4) and three experimental groups (ppm4, ppm2, ppm0)]. RNA sequencing (RNA-seq) data revealed 620 significantly differentially expressed genes (DEGs) in the control and experimental groups. By GO annotation and KEGG enrichment analysis, we found that hypoxia significantly downregulated genes associated with the immune system and inflammation, and actively maintained dissolved oxygen uptake through angiogenesis. In addition, the expression patterns of genes involved in the inhibition of cell growth and proliferation were altered under hypoxic conditions. All those mechanisms may represent a particularly important strategy for energy conservation. This study also laid the foundation for further research on the conditions associated with hypoxia in T. rubripes culture, and was helpful to the selective breeding of T. rubripes for stress tolerance.
Article
Atmospheric particulate matter (APM) emitted by iron ore processing industries has a complex composition, including diverse metallic particles and nanoparticles. Settleable APM (SePM) causes air to water cross-contamination and has recently been demonstrated to have harmful sublethal impacts on fish, eliciting stress responses, affecting the immune system, and reducing blood oxygen-carrying capacity. These findings imply potential consequences for fish aerobic performance and energy allocation, particularly in their ability to tolerate respiratory challenges such as aquatic hypoxia. To assess that potential limitation, we analyzed metabolic, cardiorespiratory, and morphological alterations after exposing tilapia, Oreochromis niloticus, to an environmentally relevant concentration of SePM (96 h) and progressive hypoxia. The contamination initiated detectable gill damage, reducing respiratory efficiency, increasing ventilatory effort, and compromising fish capacity to deal with hypoxia. Even in normoxia, the resting respiratory frequency was elevated and limited respiratory adjustments during hypoxia. SePM increased O2crit from 26 to 34% of O2 (1.84 to 2.76 mg O2·L⁻¹). Such ventilatory inefficacy implies higher ventilatory cost with relevant alterations in energy allocation. Progression in gill damage might be problematic and cause: infection, blood loss, ion imbalance, and limited cardiorespiratory performance. The contamination did not cause immediate lethality but may threaten fish populations due to limitations in physiological performance. This was the first investigation to evaluate the physiological responses of fish to hypoxia after SePM contamination. We suggest that the present level of environmental SePM deserves attention. The present results demonstrate the need for comprehensive studies on SePM effects in aquatic fauna.
Article
Full-text available
Because the stress response is so often associated with negative events, its utility has often been neglected. The selective advantages that shaped the evolution of the stress system and its regulation mechanisms is an essential foundation for understanding its costs and why HPA activity so often seems excessive.
Article
To investigate the stress response of Japanese whiting Sillago japonica associated with the capture process of sweeping trammel net, plasma cortisol levels of the fish were measured as a stress index. The result revealed that the plasma cortisol levels increased from just after capture to 24 hours, then tended to decrease in 72 hours by keeping them in a tank. In the gilled capture, changes in cortisol level were relatively similar, while for the pocketed capture, it decreased after 24 hours. Changes in plasma cortisol of mesh-cut released samples for gilled capture were relatively similar to the usual gilled capture. Hook and line stress, however, did not show any fluctuation compared to the sweeping trammel net capture. Tight contact with the mesh twine for the gilled fish during the capture process was considered to cause the higher stress of fish.
Article
The phagocytic system in goldfish was studied through intraperitoneal injection with India ink. Fish were killed at intervals ranging from 30 min to 50 days after injection. Histological research was performed on spleen, kidney, heart, liver, gills and blood.On the first day after injection, the spleen and kidney became black. Phagocytosis of carbon particles was performed from the first day after injection by the sheath cells in the sheathed artery of the spleen, by the reticuloendothelial cells throughout the haematopoietic tissue of the kidney, and by the endothelial cells in the ventricle of the heart. Carbon phagocytizing macrophages in the kidney and spleen formed aggregates within pre-existing aggregates of melano-macrophage on the fourth day after injection. Those aggregates were observed for 50 days. There was no phagocytosis of carbon particles observed in the liver and gills.Two types of mononuclear phagocytes phagocytizing carbon particles were observed in smears of blood. The large phagocytes measured between 14 and 16μ in diameter and the nucleus occupied almost half of the cell. The small phagocytes measured between 6 and 7μ in diameter and the nucleus occupied almost all of the cell. Although the small phagocytes were observed in smears during the period from the first day to the thirty-sixth day after injection, the large ones were only observed from the first day to the seventh day.The significance of the phagocytic system in fish is discussed.
Article
The salinity tolerance of the `California' Mozambique tilapia (Oreochromis mossambicus × O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l–1) tilapia hybrids were exposed to salinities from 35–95 g l–1, using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na⁺], [Cl–], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical (Na⁺, K⁺-ATPase) and morphological (number of mature, accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l–1 to 65 g l–1 with little or no change in osmoregulatory status; however, in fish exposed to 75–95 g l–1 salinity, plasma osmolality, [Na⁺], [Cl–], Na⁺, K⁺-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l–1, prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, `California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l–1 indicates they may be unable to withstand extreme salinities for extended periods of time.
We examined the effect of 2- or 24-hr confinement stress and cortisol treatment on plasma cortisol, glucose, lactate and free amino acids concentration and hepatic glycogen content and activities of certain enzymes involved in the intermediary metabolism in tilapia (Oreochromis mossambicus). Confinement of tilapia for 2 or 24 hr resulted in significantly higher plasma cortisol, glucose, lactate (2 hr), total and some of the free amino acids concentration (especially at 24 hr) and hepatic pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK; 24 hr) and lactate dehydrogenase activities (24 hr). Hepatic glycogen content was lower at 2 and 24 hr in the confined fish compared with the unstressed fish. Tilapia given cortisol implants (50 mg·kg−1 body wt) had significantly higher plasma cortisol, glucose and some of the free amino acids concentration and hepatic PEPCK and aspartate aminotransferase activities, whereas PK activity ratio was significantly lower compared with the sham group. The results suggest that glucose production 2 hr after confinement may be due to glycogenolysis, whereas the maintenance of higher glucose at 24 hr after confinement is essentially due to gluconeogenesis. Furthermore, the changes in plasma metabolites and hepatic enzyme activities with cortisol implantation suggest that cortisol plays a role in the metabolic adjustment to 24-hr confinement stress in tilapia.
Article
The utility of macrophage aggregate (MA) parameters as indicators of fish health and/or environmental stress was tested in largemouth bass (Micropterus salmoides) collected from Par Pond, a cooling reservoir for the Savannah River nuclear power plant. Initially, the effects of sex, age and season were evaluated using relative weight (Wr) in both healthy (Wr > 80) and stressed (Wr < 80) bass. There was no sex effect. A number of MA parameters, primarily hepatic and splenic no./mm2 increased linearly with age in the healthy fish. These correlations were not apparent in the stressed bass. A comparison of stressed and healthy fish of the same age showed that stressed bass had significantly more aggregates in liver and spleen as well as higher relative amounts of hepatic iron. The only seasonal difference noted in healthy bass was that fish collected in the spring had significantly less relative iron than those collected in the fall. We then tested the sensitivity of these parameters by comparing fish of similar age and Wr from two sites — the Hot Dam area which is thermally impacted and the Cold Dam arca, an unimpacted site. Fish collected from the impacted area had significantly more aggregates in the liver than those collected from the Cold Dam area. Thus, we believe these parameters to be very sensitive indicators of environmental stress.