Acute physiological stress down-regulates mRNA expressions of growth-related genes in coho salmon.

Growth and development in fish are regulated to a major extent by growth-related factors, such as liver-derived insulin-like growth factor (IGF) -1 in response to pituitary-secreted growth hormone (GH) binding to the GH receptor (GHR). Here, we report on the changes in the expressions of gh, ghr, and igf1 genes and the circulating levels of GH and IGF-1 proteins in juvenile coho salmon (Oncorhynchus kisutch) in response to handling as an acute physiological stressor. Plasma GH levels were not significantly different between stressed fish and prestressed control. Plasma IGF-1 concentrations in stressed fish 1.5 h post-stress were the same as in control fish, but levels in stressed fish decreased significantly 16 h post-stress. Real-time quantitative PCR (qPCR) analysis showed that ghr mRNA levels in pituitary, liver, and muscle decreased gradually in response to the stressor. After exposure to stress, hepatic igf1 expression transiently increased, whereas levels decreased 16 h post-stress. On the other hand, the pituitary gh mRNA level did not change in response to the stressor. These observations indicate that expression of gh, ghr, and igf1 responded differently to stress. Our results show that acute physiological stress can mainly down-regulate the expressions of growth-related genes in coho salmon in vivo. This study also suggests that a relationship between the neuroendocrine stress response and growth-related factors exists in fish.

Supraphysiological cortisol elevation alters the response of wild bluegill sunfish to subsequent stressors.

Wild fish are frequently exposed to multiple stressors, but the influence of previous or ongoing stress on an animal's subsequent response is poorly understood. Using wild-caught bluegill sunfish (Lepomis macrochirus) as a model, we used exogenous hormone implants to experimentally raise circulating cortisol in a group of fish for ∼10 days. We also maintained sham-treated and control groups of fish. We subjected all animals to a secondary stressor in the form of either a heat challenge or fasting challenge. We compared survival, body condition, and plasma-borne indicators of physiological status among cortisol-treated, sham-treated, and control groups following the secondary stressor. In order to compare short- and long-term effects of cortisol treatment, we initiated the secondary stressor either 4 or 30 days following initial cortisol treatment. Cortisol-treated fish succumbed to the fasting challenge sooner than sham-treated and control fish at both 4 and 30 days. Interestingly, cortisol-treated fish lost equilibrium sooner than sham-treated and control fish during the heat challenge when conducted at 30 days, but not at 4 days. These results demonstrate that multiple simultaneous stressors have cumulative effects on bluegill sunfish. Furthermore, these results demonstrate that supraphysiological cortisol doses alter the long-term responses of bluegill sunfish to additional challenges, even after apparent recovery. Such cumulative and long-term effects may be an important factor in mediating the response of wild animals to natural and anthropogenic stressors, and should be considered in ecological studies.

Exogenous glutathione can increase glutathione levels in tissues of rainbow trout (Oncorhynchus mykiss) through extracellular breakdown and intracellular synthesis.

Glutathione (GSH) is an important intracellular antioxidant involved in numerous cellular pathways. However, little is known about the transport of GSH into fish tissues. To determine whether fish tissues took up GSH by extracellular breakdown and intracellular synthesis or by direct cellular transport, we injected rainbow trout (Oncorhynchus mykiss) with exogenous GSH along with blockers of GSH breakdown and synthesis. Exogenous GSH increased GSH levels to the greatest degree in the cells of the posterior kidney, followed by the liver. Exogenous GSH inconsistently increased liver GSH levels independent of GSH synthesis, although this may have been due to disruption of gradient-dependent GSH export, and not necessarily to intact uptake of GSH. The cells of the posterior kidney, liver and gill took up GSH by extracellular breakdown and intracellular synthesis. This indicates that, unlike mammalian tissues, normal cellular GSH levels in fish are not sufficient to inhibit additional GSH synthesis. This may lend flexibility to the GSH system in fish, where levels of GSH may rapidly increase in response to an increased supply of amino acids, or during times of high demand, without increasing synthesis enzymes.

Physiological stress responses in the warm-water fish matrinxã (Brycon amazonicus ) subjected to a sudden cold shock / Respostas fisiológicas ao estresse do peixe de águas tépidas matrinxã (Brycon amazonicus ) submetido à queda brusca de temperatura

The present work evaluated several aspects of the generalized stress response [endocrine (cortisol), metabolic (glucose), hematologic (hematocrit and hemoglobin) and cellular (HSP70)] in the Amazonian warm-water fish matrinxã (Brycon amazonicus ) subjected to an acute cold shock. This species farming has been done in South America, and growth and feed conversion rates have been interesting. However, in subtropical areas of Brazil, where the water temperature can rapidly change, high rates of matrinxã mortality have been associated with abrupt decrease in the water temperature. Thus, we subjected matrinxã to a sudden cold shock by transferring the fish directly to tanks in which the water temperature was 10ºC below the initial conditions (cold shock from 28ºC to 18ºC). After 1h the fish were returned to the original tanks (28ºC). The handling associated with tank transfer was also imposed on control groups (not exposed to cold shock). While exposure to cold shock did not alter the measured physiological conditions within 1h, fish returned to the ambient condition (water at 28º C) significantly increased plasma cortisol and glucose levels. Exposure to cold shock and return to the warm water did not affect HSP70 levels. The increased plasma cortisol and glucose levels after returning the fish to warm water suggest that matrinxã requires cortisol and glucose for adaptation to increased temperature.

O presente trabalho avaliou as principais respostas fisiológicas e celulares [endócrino (cortisol), metabólico (glicose), hematológico (hematócrito and hemoglobina) e celular (HSP70)] ao estresse de um peixe de águas tépidas, o matrinxã (Brycon amazonicus ), quando submetido a um choque térmico frio abrupto. Essa espécie vem sendo amplamente cultivada na América do Sul por apresentar excelentes índices zootécnicos de crescimento e conversão alimentar. Entretanto, os produtores rurais encontram limitações no manejo do matrinxã, quando criado em regiões mais frias que sua região de origem, a Bacia Amazônica. Assim, o matrinxã foi submetido a um choque frio através da transferência direta dos peixes para tanques com água fria a 18ºC. Após 1h, esses peixes retornaram a suas caixas de origem a 28ºC. O manuseio de peixes necessário para conduzir o choque térmico experimental foi também imposto aos grupos controle, sendo, entretanto, evitada a água fria. O matrinxã demonstrou claros sinais de estresse fisiológico durante os procedimentos experimentais. Porém, essas respostas não foram associadas ao choque frio, mas sim ao choque quente por ocasião da volta dos peixes para as caixas de origem. As respostas primárias e secundárias de estresse foram evidentes através das análises plasmáticas de cortisol e glicose. Já o hematócrito, a hemoglobina e as expressões da proteína de estresse, HSP70, não foram afetadas. Nossos resultados sustentam que o matrinxã falhou em responder ao choque térmico frio, mas não ao choque térmico quente, que é um estressor evidentemente associado à origem natural dessa espécie de águas de elevadas temperaturas.

Impairment of the stress response in matrinxã juveniles (Brycon amazonicus) exposed to low concentrations of phenol.

In this study we measured plasma cortisol, plasma glucose, plasma sodium and potassium, and liver and gill hsp70 levels in juvenile matrinxã (Brycon amazonicus) subjected to a 96 h exposure to phenol (0, 0.2, and 2.0 ppm), and the effect of this exposure on their ability to respond to a subsequent handling stress. Fish were sampled prior to initiation of exposure and 96 h, and at 1, 6, 12, and 24 h post-handling stress. During the 96 h exposure, plasma cortisol and glucose levels remained unchanged in all treatments. While plasma sodium levels were significantly reduced in all groups, plasma potassium levels only decreased in fish exposed to 0 and 0.2 ppm of phenol. Liver hsp70 levels decreased significantly at 96 h in fish exposed to 2.0 ppm of phenol. All groups, except fish exposed to 0.2 ppm of phenol, were able to increase plasma cortisol and glucose levels after handling stress. Fish exposed to 2.0 ppm of phenol showed decreased gill and liver hsp70 levels after the handling stress. Our data suggest that exposure to phenol may compromise the ability of matrinxã to elicit physiological responses to a subsequent stressor.

The welfare of fish.

Our interactions with fish cover a wide range of activities including enjoying them as pets to consuming them as food. I propose that we confine the consideration of the welfare of fish to their physiology, and not join the discussion on whether fish can feel pain and suffering, as humans. A significant proportion of the papers on animal welfare center on whether non-human animals can feel pain, and suffer as humans. This is a question that never can be answered unequivocally. The premise of the present paper is that we have an ethical responsibility to respect the life and wellbeing of all organisms. Thus, we should concentrate on the behavioural, physiological, and cellular indicators of their well-being and attempt to minimize a state of stress in the animals that we have in our care or influence.

The glutathione antioxidant system is enhanced in growth hormone transgenic coho salmon (Oncorhynchus kisutch).

Insertion of a growth hormone (GH) transgene in coho salmon results in accelerated growth, and increased feeding and metabolic rates. Whether other physiological systems within the fish are adjusted to this accelerated growth has not been well explored. We examined the effects of a GH transgene and feeding level on the antioxidant glutathione and its associated enzymes in various tissues of coho salmon. When transgenic and control salmon were fed to satiation, transgenic fish had increased tissue glutathione, increased hepatic glutathione reductase activity, decreased hepatic activity of the glutathione synthesis enzyme gamma-glutamylcysteine synthetase, and increased intestinal activity of the glutathione catabolic enzyme gamma-glutamyltranspeptidase. However, these differences were mostly abolished by ration restriction and fasting, indicating that upregulation of the glutathione antioxidant system was due to accelerated growth, and not to intrinsic effects of the transgene. Increased food intake and ability to digest potential dietary glutathione, and not increased activity of glutathione synthesis enzymes, likely contributed to the higher levels of glutathione in transgenic fish. Components of the glutathione antioxidant system are likely upregulated to combat potentially higher reactive oxygen species production from increased metabolic rates in GH transgenic salmon.

Effects of the natural tidal cycle and artificial temperature cycling on Hsp levels in the tidepool sculpin Oligocottus maculosus.

The rocky intertidal zone is characterized by a predictable cycle of environmental change cued by the ebb and flow of the tides. Tidepools are thus an excellent environment in which to determine whether predictability of environmental change can entrain an endogenous rhythmicity in heat shock protein (Hsp) levels. In this study, we monitored changes in Hsp mRNA and protein levels that occurred over the tidal cycle in tidepool sculpins and investigated whether there was an endogenous tidal rhythm in Hsp expression that persisted once the sculpins were transferred to a stable environment. Fluctuations in the tidepool environment increased hsc70, hsp70, and hsp90 mRNA levels, which translated into increased Hsc/Hsp70 and Hsp90 protein levels; however, this was not due to an endogenous tidal rhythm in Hsp levels because sculpins held under constant conditions did not show any rhythmicity in the expression of these genes. By exposing sculpins to an artificial temperature cycling regime that mimicked the temperature changes of a mid-intertidal pool, we were able to account for the direct role of temperature in regulating Hsp expression. However, there are additional extrinsic factors that likely integrate with temperature and result in differences between the hsp induction profiles that were observed in sculpins inhabiting their natural environment and those in cycling conditions in the laboratory.

Low levels of environmental ammonia increase susceptibility to disease in Chinook salmon smolts.

Ammonia criteria are established using data from standardized toxicity tests involving healthy animals. Both intrinsic and extrinsic environmental changes affect the immune system, but few toxicity studies consider the overall impact on this system and potential changes in resistance to infection. To investigate the effects of subacute levels of ammonia in coastal waters on physiological and immunological systems of fish, juvenile Chinook salmon were maintained in seawater (10 degrees C, pH 7.8) and exposed to two concentrations of ammonia, 2.5 and 10 mg/L total nitrogen. Both test levels resulted in increased internal levels of ammonia in the fish. Neither treatment level affected feeding rates. Over a time course of 10 d, numerous significant effects were observed. White blood cell counts changed significantly, as did respiratory burst activity, plasma lysozyme activity, and plasma glucose concentration in both treatments compared to controls. In an experimental infection with Vibrio anguillarum, fish previously exposed to subacute levels of ammonia were more susceptible to pathogenic challenge. The findings of this study indicate that a more thorough investigation into the effects of environmental ammonia on fish populations in coastal waters should be undertaken and the current environmental standards reassessed.

Effects of clove oil on the stress response of matrinxã (Brycon cephalus) subjected to transport

O transporte de peixes vivos é certamente um dos principais estímulos adversos à homeostase dos peixes nas condições de criação em cativeiro. O presente trabalho mensurou o estresse do matrinxã (Brycon cephalus), quando submetido ao transporte em sacos plásticos, bem como avaliou os efeitos do uso do anestésico óleo de cravo nessa etapa do manejo. Foram testadas as concentrações de 0, 1, 5 e 10 mg/L de óleo de cravo em bolsas plásticas preenchidas com água e oxigênio, de acordo com as práticas comumente utilizadas no Brasil. O óleo de cravo reduziu algumas das principais respostas ao estresse (cortisol, glicose plasmática e íons) mensuradas. O alto gasto de energia para o matrinxã tolerar o transporte foi evidenciado pela diminuição dos valores de glicogênio hepático. Os resultados sugerem que o óleo de cravo em concentração de 5 mg/L pode atenuar as principais respostas de estresse do matrinxã durante o transporte.

Cross-tolerance in the tidepool sculpin: the role of heat shock proteins.

Cross-tolerance, or the ability of one stressor to transiently increase tolerance to a second heterologous stressor, is thought to involve the induction of heat shock proteins (Hsp). We thus investigated the boundaries of cross-tolerance in tidepool sculpins (Oligocottus maculosus) and their relationship to Hsp70 levels. Survival of sculpins exposed to severe osmotic (90 ppt, 2 h) and hypoxic (0.33 mg O(2)/L, 2 h) stressors increased from 68% to 96%, and from 47% to 76%, respectively, following a +12 degrees C heat shock. The magnitude of this heat shock was critical for protection. A +10 degrees C heat shock did not confer cross-tolerance, while a +15 degrees C heat shock was deleterious. Sculpins required between 8 and 48 h of recovery following the +12 degrees C heat shock to develop cross-tolerance. There was no association between Hsp70 levels before the onset of the secondary stressor and cross-tolerance. However, branchial Hsp70 levels following osmotic shock were highly correlated with the time frame of cross-tolerance. Thus, Hsp70 induction by the priming stressor may be less important than the ability of the cell to mount an Hsp response to subsequent stressors. The time frame of cross-tolerance is similar to the interval between low tides, suggesting the possible relevance of this response in nature.

Are hsps suitable for indicating stressed states in fish?

In response to most stressors, fish will elicit a generalized physiological stress response, which involves the activation of the hypothalamic-pituitary-interrenal axis (HPI). As in other vertebrates, this generalized stress response comprises physiological responses that are common to a wide range of environmental, physical and biological stressors. Recently, several families of heat shock proteins (hsps) have been proposed as indicators of a generalized stress response at the cellular level. Recent findings that hsp levels, in various fish tissues, respond to a wide range of stressors have supported the use of these proteins as indicators of stressed states in fish. However, the cellular stress response can vary, for example, according to tissue, hsp family and type of stressor. This brief overview of these responses in fish asks the question of whether changes in levels and families of hsps can be used as a suitable indicator of stressed states in fish. By casting this question in the context of the well-established generalized physiological stress response in fish, we argue that the use of hsps as indicators of stressed states in fish in general is premature.

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus.

Effects of environmental salinity and 17alpha-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3-5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.

Heat shock protein genes and their functional significance in fish.

Despite decades of intensive investigation, important questions remain regarding the functional, ecological, and evolutionary roles of heat shock proteins. In this paper, we discuss the utility of fish as a model system to address these questions, and review the relevant studies of heat shock protein genes and the regulation of their expression in fish. Although molecular studies of the heat shock proteins in fish are still in their early descriptive phase, data are rapidly being collected. More is known about the biotic and abiotic factors regulating heat shock proteins. We briefly review these studies and focus on the role of heat shock proteins in development, their regulation by the endocrine system, and their importance in fish in nature. Functional genomics approaches will provide the tools necessary to gain a comprehensive understanding of the significance of heat shock proteins in the cellular stress response, in the physiological processes at higher levels of organization, and in the whole animal in its natural environment.