Morphologic Effects of the Stress Response in Fish

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Yet few studies have addressed stress as a potential confounding factor for bioassays that use fish as test subjects. Common stressors encountered by captive ...

Morphologic Effects of the Stress Response in Fish

Claudia Harper and Jeffrey C. Wolf

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 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. Key Words: contaminant; crowding; fish; handling; histology; nutrition; temperature; salinity; stress



n 1936 a scientist named Hans Selye, upon observing effects of noxious stimuli in laboratory animals, coined the term “stress” and defined it as “the non-specific response of the body to any demand for change” (Selye 1936,

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 [email protected]

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32). Subsequent researchers have proposed a variety of alternate definitions, 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 example, exposure to cold temperatures (stimulus) can make an individual feel cold (awareness) and evoke shivering (response); regarded independently, each of these elements might be considered stress. As one frustrated scientist reportedly 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 indicate stressful stimuli and “stress responses” to designate the reactions to such stimuli. The purpose of this article is to review morphologic effects of various stressors in fish as determined by gross or histopathologic investigation. In addition, descriptions of both tissue-specific and non-tissue-specific 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., ambient temperature, oxygen supply), insufficient food availability, inadequate refuge from sunlight or predators, and the demands of social interactions such as territorial disputes. Other stressors are unique to certain animal groups or habitats. As compared to terrestrial inhabitants, fish 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 fish include fluctuations in water salinity, pH, hardness, alkalinity, dissolved solids, water level or current, and exposure to waterborne pathogens or toxicants. Fish reared in confinement systems often experience further pressures of crowding, handling, suboptimal nutrition, and nitrogenous waste accumulation. 387

Evidence indicates that certain stress responses are well conserved evolutionarily. In terms of behavior, an obvious example is the instinctive urge to fight or flee when faced with an adverse stressor such as predation. Many physiological responses to stressors are also remarkably comparable among taxonomically diverse animals. For instance, common among all vertebrates is the stressor-induced secretion of adrenergic and glucocorticoid hormones; the latter especially is considered a hallmark of the stress response (Nesse and Young 2000). Although fish lack adrenal glands per se, analogous production and release of adrenal cortical and medullary hormones occur in the interrenal cells and chromaffin tissues, respectively, both of which are typically located in the piscine anterior kidney. As in the case of so-called higher vertebrates, the secretion of stress-related hormones in fish can be a double-edged sword. The activities of these hormones are clearly beneficial when acute action and its consequences take priority, as they elicit a heightened state of alertness, increase blood pressure and respiration, promote hepatic glycogen catabolism to provide a source of energy via glucose, and limit excessive tissue damage from inflammatory reactions to trauma or illness (Nesse and Young 2000). However, hormonal stress responses that overcompensate or persist can also have negative effects, such as immune suppression, depletion of energy reserves, muscle breakdown, and, in fish, interference with osmoregulation as a result of altered mineral metabolism (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 experimental setting. First, such responses are not “all or nothing” 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 manifestations (e.g., decreased reproductive performance) to patent signs of disease (e.g., life-threatening microorganism infections) (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 contribute 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 fish 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 difficult to distinguish from effects of the stressor itself (Selye 1955); this represents an important obstacle in endocrine disruption research, in which it is necessary to differentiate the particular effects of hormonally active substances from their concomitant ability to contribute to the stress response (Norris 2000). 388

To date, methods for evaluating stress responses in fish have involved a variety of endpoints: • whole body or organ weight measurements (e.g., condition factor, hepatosomatic index, and gonadosomatic index) (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 proteins) (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 fish steroids in water (Scott and Ellis 2007); and • macroscopic and microscopic anatomy (numerous references cited in the following text). As a research tool, the histopathologic evaluation of whole body sections from small fish species offers numerous advantages, including the ability to observe a wide variety of organ systems in relatively few tissue sections, the ability to identify 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 detect 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 number of studies have specifically addressed the effects of such responses on tissue histomorphology. For example, despite documented stressor-induced alterations of reproductive system endpoints (Cleary et al. 2002; Contreras-Sánchez et al. 1998), there has been little effort to determine potential histopathologic effects of the stress response (e.g., as modeled by cortisol administration) on fish gonads or gonadal ducts; such effects might include increased germ cell degeneration in the ovary (oocyte atresia) and/or testis. Such findings would be significant because those same types of changes are often regarded as prima facie evidence of endocrine disruption (Heiden et al. 2006; Leino et al. 2005; Rasmussen et al. 2005). Seasonal changes also are known to affect fish gonads and lead to morphological changes (Abe and Munehara 2007).

Fish-Specific Stressors There are roughly 30,000 known species of fish, and both wild and captive fishes occupy a remarkably diverse array of habitats. Accordingly, environmental conditions that might be optimal for one species are inherently stressful for another. Given the number of potential stressors, and the fact that fish may be exposed to multiple stressors simultaneously, the range of potential stress-inducing situations is almost limitless. This section provides brief descriptions of commonly encountered stressors and the anatomic sites in which corresponding morphologic effects tend to occur. ILAR Journal

Specific histopathologic findings are described in more detail 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 fish, but wild fish may also experience these disturbances, for example through catch and release programs in recreational fisheries. Procedures that can intensify the stress response in aquacultured fish include sorting, grading, and vaccine administration (Burgess and Coss 1982). Additional stressful sequelae include crowding, hypoxia, physical trauma, aftereffects of anesthetics or sedatives, and barometric disturbance in fish harvested at considerable depth. Evidence that these stimuli are intrinsically stressful is provided by experiments that have documented marked increases in blood cortisol and/or glucose levels in fish following deliberate handling and transport (Acerete et al. 2004; Barton 2002; Hosoya et al. 2007). There may be some benefit to sedating fish before transport in order to mitigate shipping stress. In a study in which channel catfish (Ictalurus punctatus) were subjected to stressors such as confinement, high ammonia, and oxygen depletion, sedation resulted in lower cortisol elevations than those observed in control fish (Small 2004). But the magnitude of the stress response to netting, transport, and handling varies considerably among species and, typical of stress responses in fish, 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 fish experience these stressors, there has been very little investigation of potential histomorphologic consequences. For example, although anecdotal observations suggest that fish may suffer microscopically evident muscle degeneration (rhabdomyolysis) as a consequence of collection (capture myopathy), experiments have not been conducted to confirm this causal relationship.

Crowding For captive fish, appropriate stocking density varies greatly according to the species, housing system, and available resources. Overcrowding may be accompanied by additional stressors such as poor water quality, exposure to organic wastes, and conspecific aggression and predation. Gilthead seabream (Sparus aurata L.) experienced significant rapid increases in blood cortisol and glucose following short-term crowding (Ortuño et al. 2001), and similar results were observed 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 confinement were attributed to glycogenolysis, whereas in fish confined for 24 hours gluconeogenesis was considered the primary mechanism for glucose elevations (Vijayan et al. 1997). Although morphologic changes were not the focus of these experiVolume 50, Number 4


ments, it is reasonable to surmise that certain durations of confinement stress might therefore manifest in histopathologic findings such as decreased hepatocellular vacuolation (especially in cultured fish) and muscle atrophy. Burgess and Coss (1982) examined histologic specimens from adult jewel fish (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 fluctuations that preclude acclimation or inappropriate water temperature (beyond the high or low range of tolerance). A rapid temperature decrease limits a fish’s ability to produce antibodies integral to an immediate immune response, and a delay in the immune response may enable pathogens to colonize, reproduce, and establish an infection. Very cold temperatures may inactivate defensive functions of nonspecific 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 temperature changes over time (Kurata et al. 1995). Hyperthermia has been used experimentally as a stressor in challenge studies 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 environments, but hypoxia can also occur when fish are shipped in insufficiently aerated containers, for example. The decrease in oxygen availability to tissues can lead to necrotic or apoptotic lesions in organs (Geng 2003; van der Meer et al. 2005). In channel catfish, experimentally induced sublethal hypoxia was responsible for histopathologically evident necrosis, hyperemia (vascular congestion), edema, hemorrhage, hyperplasia, 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 reasonably argued that such lesions formed as a specific reaction to acute localized oxygen deprivation rather than to stress per se, it is plausible that stress contributed to the response on some level. Some teleost fish, frogs, turtles, snakes, and insects have the capacity to tolerate or adapt to hypoxia (van der Meer et al. 2005). For instance, zebrafish (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 389

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 fish species, including the hearts of zebrafish 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 platyfish (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 fishes 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 fish are under continuous pressure to conserve salts, whereas the reverse is true for marine species, which must conserve water (Greenwell et al. 2003). Among fishes 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 fluctuate with tides, season, or evaporation from surface waters. Few studies have investigated potential morphologic effects of salinity as the sole stressor. An experiment to assess optimal stocking densities for sea bass (Dicentrarchus labrax) fingerlings applied hypersalinity as a stressor along with temperature modifications (Via et al. 1998). But an experiment that specifically evaluated the tolerance of hybrid tilapia (Oreochromis mossambicus × O. urolepis hornorum) to hypersaline water found that the primary morphologic indicators of hypersaline stress, and the most sensitive of several endpoints tested, were ultrastructural changes in the gills (Sardella et al. 2004). In anadromous fish such as salmon, physiological changes associated with smoltification (the metamorphic transformation 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 simplified classification system, malnutrition can be categorized as disorders that result from either (1) an insufficiency or overabundance of nutrients or (2) relative nutrient imbalances. Factors that typically contribute to malnutrition in wild fish include depletion of species-appropriate food sources or components (e.g., vitamins, minerals), heightened competition for available food resources, and inappetence due to disease. Captive fish often endure the additional challenge of suboptimal feed formulation, usually because the precise nutritional requirements for the fish species of interest have not been determined, a suitable diet can390

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 difficult to separate stress responses from the direct effects of malnutrition in terms of morphologic consequences. For example, 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 stressinduced corticosteroid-mediated glycogenolysis (Barton and Schreck 1987; Vijayan et al. 1997). Furthermore, food deprivation can lead to reduced stress resistance, as was the outcome 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 influence the stress response, as higher blood cortisol concentrations in rainbow trout were associated with dietary variations of vitamin E, vitamin C, and highly unsaturated fatty acids (Trenzado et al. 2008). In such cases it may be difficult to discriminate the stressor from the stress response; for example, interrenal ascorbic acid concentrations decreased in rainbow trout and coho salmon (Oncorhynchus kisutch) that were subjected to nonspecific stress (Wedemeyer 1969).

Contaminants Fish have been exposed, either intentionally or unintentionally, to a vast array of chemical and particulate contaminants, of both natural and man-made origin. Examples include pharmaceuticals, agricultural chemicals, manufacturing byproducts, animal and human waste materials, mining effluents, and substances released as a consequence of natural disasters such as fires. Arguably, at sufficient concentration, almost any contaminant is capable of inducing a stress response. 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. However, 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, insufficient 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 number of fish species. Acute ammonia toxicity can cause an assortment of clinical signs in fish, 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 injection) (Randall and Tsui 2002; Spencer et al. 2008). ILAR Journal

Ammonia exposure has been associated with morphologic findings in a variety of fish 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 fish that had suffered from neurological dysfunction (Daoust and Ferguson 1984). In addition to the gills, ammonia-related lesions have been reported in the liver, kidney, intestine, and ovary of fish (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 chromaffin 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 include hyperglycemia due to enhanced glycogenolysis and gluconeogenesis, vasodilation of arteries in gill filaments, increased cardiac stroke volume, and immune function depression (Gratzek and Reinert 1984). The first two phases are considered adaptive and enable fish to adjust to stressors and maintain homeostasis. In contrast, tertiary responses involve systemic changes in which animals may become incapable 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, morphologic indications of stress also exist in tissue types that are less routinely examined.

Gills Given the relative fragility of the gills compared to other surface tissues, and the fact that they are continually exposed to the fish’s external environment, it is remarkable that these structures are able to survive and compensate for the chemical and physical assaults to which they are invariably subjected. 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 fishes have developed intriguing adaptive stress response mechanisms. For example, the gills of Crucian carp exhibit a reversible morphological reaction to decreased oxygen availability (Sollid et al. 2003), thanks to a unique anatomic feature: under normal ambient oxygen concentrations, the gills lack protruding secondary lamellae (typically the primary sites of gas exchange in other fishes); instead, the secondary lamellae are embedded in a cell mass that, by deVolume 50, Number 4


Figure 1 Nonspecific stress response in the gills of adult Atlantic salmon (Salmo salar L.). (A) Normal gill (two adjacent filaments). (B) Findings associated with several types of stressors; the most prominent changes are mucus cell hyperplasia (arrow) and epithelial lifting (arrowhead). Bar = 50 microns.

sign, decreases the respiratory surface area. Under hypoxic conditions, this cell mass recedes due to the combined effects of increased apoptosis and diminished cell proliferation, and as it shrinks it exposes the underlying lamellae, thus increasing the overall surface area of the gills. This adaptation may have evolved to reduce water and ion flux under normoxic conditions and thus conserve energy for osmoregulation. Similarly, in various African cichlid fish exposure to long-term hypoxia resulted in elongation of branchial filaments 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 catfish exposed to varying degrees of sublethal hypoxia exhibited a suite of nonspecific, histologically evident changes likely to interfere with respiratory gas exchange, such as gill epithelial hypertrophy and hyperplasia, goblet cell proliferation with increased mucus secretion, hemorrhage, edema, and telangiectasis (Scott and Rogers 1980). 391

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 acidbase regulation; Perry 1998) occurred in hybrid tilapia exposed 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 nonspecific responses such as lamellar thickening, mucus cell hyperplasia and hypertrophy, epithelial cell lifting, leukocyte infiltration, hyperemia, hemorrhage, chloride cell proliferation, secondary lamellar fusion, and telangiectasis (Benli et al. 2008; Lease et al. 2003; Spencer et al. 2008). After an investigation of the combined effects of ammonia and elevated pH in Lost River suckers, Lease and colleagues (2003) concluded that structural gill changes were more sensitive than other traditional assays for detecting ammonia toxicity. An earlier study recorded similar types of morphologic findings in wild freshwater fish exposed to a mixture of known and unknown contaminants (Teh et al. 1997). Gill lesions in that study included hyperplastic mucous and chloride cells, deformed branchial cartilages, severe 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 response. But apparently this is not necessarily the case, as one study has demonstrated that social stress did not lead to chloride 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 efficient 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 hepatocellular vacuolation and tinctorial staining characteristics (Wolf and Wolfe 2005). Decreased vacuolation can result from loss of cytoplasmic glycogen and/or lipid caused by insufficient energy intake relative to need and/or glucocorticoid-induced glycogenolysis. Conversely, increased hepatocellular vacuolation 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 cytoplasmic vacuolation were not features of hypoxia in channel catfish, which instead showed hepatic necrosis and hemorrhage as well as splenic changes such as edema, hyperemia, and necrosis. Chronic histopathologic changes such as se392

vere hepatic lipidosis, lymphoid cell depletion, vascular congestion, and reticuloendothelial cell necrosis (in the spleen) were evident in the livers and spleens of wild freshwater fish exposed to mixed contaminants (Teh et al. 1997). The added presence of a number of preneoplastic and neoplastic proliferative lesions in those fish 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 protective physical barrier that is important in terms of both osmoregulation and pathogen defense. But fish skin is susceptible to damage from handling, fighting, physical trauma, predation, 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 defenses, via corticosteroid-mediated immunosuppression or other stress-related immunosuppressive factors (Choi et al. 2007; Harris et al. 2000; Kent and Hedrick 1987). Although fish skin has not been reported extensively as a stress response target, dermal ulceration was the chief finding in a series of studies in which striped bass (Morone saxatilis) and striped bass hybrids were exposed to acute confinement stress (Noga et al. 1998; Udomkusonsri et al. 2004). Associated histopathologic lesions, in addition to rapidly occurring epithelial erosions and ulcers that primarily affected the fins, 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 fish reproductive system due to various stressors (capture, handling, crowding, hypoxia, tank draining, 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 effects of such stressors in the gonads or genital ducts. One study found retarded oocyte maturation in common carp exposed 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 morphometric counting of ovarian and testicular germ cells in histologic sections) in both male and female fish (Thomas et al. 2007). There are even fewer reports of stress responses that involve the fish urinary tract. Examples include hypoxiainduced hemorrhage, glomerular congestion, and edema in the posterior kidneys of channel catfish (Scott and Rogers 1980), and congestion in Nile tilapia exposed to sublethal concentrations of ammonia (Benli et al. 2008). ILAR Journal

Nervous and Sensory Systems Routine diagnostic examinations or experimental investigations involving fish tissues tend to include sampling of the brain and spinal cord less frequently than for other organs. Although inflammation and endoparasitism of the central nervous system are often readily recognizable in standard histologic sections, more subtle types of changes are not always easily appreciated. For example, in a series of experiments in which jewel fish were exposed to chronic crowding stress, special histologic staining and morphometric techniques were required in order to determine that, compared to controls, crowded fish had structural nerve cell alterations (both qualitative and quantitative) in the optic tectum, a major area of the brain concerned with processing and integrating 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 detection of stress-related changes in the eyes may also require detailed examination. For example, findings in perinatal platyfish subjected to hypoxic conditions included central corneal thinning, hyperplasia of corneal endothelial cells, lens fiber derangement, and apoptotic cells in the retina (Chan et al. 2007). Perhaps more obvious were the corneal ulcerations induced by acute confinement stress in hybrid striped bass (Morone saxatilis × M. chrysops) (Udomkusonsri et al. 2004).

in many different fish tissues including blood vessels in hypoxia-tolerant fish (Cossins et al. 2009).

Multiorgan and Systemic Stress Responses Systemic stress responses include alterations (often decreases) in body condition and/or organ weights, with corresponding 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 formation of histologically evident pigmented macrophage aggregates (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

Cardiovascular System Histologically evident changes in the hearts of adult zebrafish and Lake Victoria cichlids (Haplochromis piceatus) subjected to chronic hypoxia included reduced ventricular outflow tracts and reduced lacunae surrounding trabeculae (Marques et al. 2008). Quantitation of myocyte nuclei in both species also revealed that, relative to controls, hypoxic fish had increased numbers of nuclei per unit area. Occasionally, microscopic examinations of blood smears can reveal morphologic evidence of stress that would be difficult to detect in tissue sections. For example, two classic hematological manifestations of the stress response in mammals, neutrophilia and lymphopenia, were triggered in Nile tilapia by acute hypoxia followed by reperfusion (Choi et al. 2007). Although a description of hematological changes associated with stress is outside the scope of this article, many publications clearly indicate that stressors such as handling, crowding, capture, restraint, hypoxia, anesthesia, air exposure, and sampling technique can affect fish 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 fish; for example, recent evidence indicates that unique types of myoglobin are present Volume 50, Number 4


Figure 2 Histomorphologic effects of chronic starvation in adult female Japanese medaka (Oryzias latipes). Images (A), (C), and (E) are from a well-nourished fish; (B), (D), and (F) are from a fish 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 fiber size. (C) Normal liver; arrowheads indicate moderate hepatocyte vacuolation consistent 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 abundant 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. 393

Figure 3 Pigmented macrophage aggregates (PMA). In this photomicrograph of the anterior kidney from a striped bass (Morone saxatilis), PMA are compared to an early stage granuloma (EG) and a late stage granuloma (LG), both of which are an inflammatory response to a mycobacterial infection. The inset illustrates a PMA at higher magnification. Although it is likely that a few of the macrophages in this PMA also contain mycobacteria, in most cases PMA formation occurs secondary to noninfectious causes. Bar = 50 microns.

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 inflammation, which are more typically a response to microbial infection, for example. Some of the many functions attributed to PMA include sequestration of cell breakdown products, recycling and storage of iron, antigen presentation, and detoxification of exogenous 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 fish age, but reports indicate that proliferation of these structures may also occur as a nonspecific 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” remains nebulous. First, the interplay between stressors and stress responses is highly complex, and some stress responses may themselves function as stressors, and vice versa. Second, there are few, if any, pathognomonic stress responses (the nonspecific nature of stress responses is in keeping with Selye’s original definition). A third explanation concerns the tendency of researchers to use the term “stress” to indicate 394

almost any type of adverse condition that a fish might encounter or any form of outcome. Purists may argue, somewhat justifiably, 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 stressful, but the associated morphologic effects may actually reflect 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 defined, to confound a study’s results. Failure 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 attributable to a particular stressor under specified conditions. Further challenge studies of fish may enhance understanding of stress and its effects in fish through the administration of glucocorticoid or adrenergic hormones, heat shock proteins, or other types of mediators not yet identified.

Acknowledgments Funding for this project was provided in part by Experimental Pathology Laboratories Inc., in Sterling, Virginia.

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